Methods and systems for rapid detection of cronobacter using infectious agents

Abstract

To provide methods and systems for rapid detection of Cronobacter using infectious agents.SOLUTION: Disclosed herein are methods and systems for rapid detection of microorganisms such as Cronobacter spp. in a sample. A genetically modified bacteriophage is also disclosed which comprises an indicator gene in the late gene region. The specificity of a bacteriophage, such as a Cronobacter-specific bacteriophage, allows detection of a specific microorganism, such as Cronobacter spp., and an indicator signal may be amplified to optimize assay sensitivity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 62 / 616,956, filed January 12, 2018; 62 / 628,616, filed February 9, 2018; and 62 / 661,739, filed April 24, 2019. The disclosures of U.S. Patent Application Nos. 13 / 773,339, 14 / 625,481, 15 / 263,619, 15 / 409,258 and U.S. Provisional Patent Application Nos. 62 / 616,956, 62 / 628,616, and 62 / 661,739 are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to compositions, methods, systems and kits for the detection of microorganisms using infectious agents. [Background technology]

[0003] background There is strong importance in improving the speed and sensitivity for the detection of bacteria, viruses, and other microorganisms in biological, food, water, and clinical samples. Microbial pathogens can cause substantial morbidity in humans and domestic animals, as well as enormous economic losses. In addition, microbial detection is a high priority for the U.S. Food and Drug Administration (FDA) and the Centers for Disease Control and Prevention (CDC), as well as the U.S. Department of Agriculture (USDA), in light of the outbreaks of life-threatening or fatal diseases caused by the ingestion of food contaminated with certain microorganisms (e.g., Cronobacter spp., Salmonella spp., Listeria spp., or Staphylococcus spp.).

[0004] Traditional microbiological tests for detecting bacteria rely on nonselective and selective enrichment cultures, followed by plating on selective media and further testing to confirm suspect colonies. Such procedures can require several days. Various rapid methods have been investigated and introduced into practice to reduce the time requirements. However, these methods have drawbacks. For example, direct immunoassays or techniques involving gene probes generally require an overnight enrichment step to achieve adequate sensitivity. Polymerase chain reaction (PCR) tests also involve an amplification step and are therefore capable of both very high sensitivity and selectivity; however, economically, the sample size that can be subjected to PCR testing is limited. With dilute bacterial suspensions, most small subsamples are cell-free and therefore still require purification and / or lengthy enrichment steps.

[0005] The time required for traditional biological enrichment is dictated by the growth rate of the target bacterial population in the sample, by the effect of the sample matrix, and by the sensitivity required. In practice, most sensitive methods use overnight incubation, taking approximately 24 hours overall. Due to the time required for culture, these methods can take up to three days, depending on the organism to be identified and the source of the sample. This lag time is generally determined by the amount of time spent in contaminated food, water (or other product). It is inappropriate to use a bacterial pathogen in water, food, and clinical samples because the pathogen may have already made its way into livestock or humans. Furthermore, the rise of antibiotic-resistant bacteria and biodefense concerns has made rapid identification of bacterial pathogens in water, food, and clinical samples a critical priority worldwide.

[0006] Thus, there is a need for faster, simpler, and more sensitive detection and identification of microorganisms, such as bacteria and other potentially pathogenic microorganisms. Summary of the Invention [Means for solving the problem]

[0007] Abstract Embodiments of the present invention include compositions, methods, systems and kits for the detection of microorganisms, such as Cronobacter spp. The present invention can be embodied in a variety of ways.

[0008] In some aspects, the invention includes a recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome. In some embodiments, the recombinant bacteriophage is a genetically modified Cronobacter-specific bacteriophage genome. In certain embodiments, the recombinant bacteriophage comprises a genetically modified Cronobacter-specific bacteriophage genome derived from a bacteriophage that specifically recognizes Cronobacter spp. (formerly classified as Enterobacter sakazakii). In some embodiments, the bacteriophage used to prepare the recombinant bacteriophage is a bacteriophage that specifically recognizes one or more Cronobacter In one embodiment, the recombinant bacteriophage is capable of distinguishing Cronobacter spp. in the presence of other types of bacteria.

[0009] In some embodiments of recombinant indicator bacteriophages, the indicator gene may be codon-optimized and encode a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. Some recombinant bacteriophages further comprise an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site. In some embodiments, the indicator gene is a luciferase gene. The luciferase gene may be a naturally occurring gene, such as an Oplophorus luciferase gene, a firefly luciferase gene, a Lucia luciferase gene, or a Renilla luciferase gene, or the luciferase gene may be a genetically engineered gene, such as NANOLUC®.

[0010] Also disclosed herein is a method for preparing a recombinant indicator bacteriophage. Some embodiments include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid / vector and the bacteriophage genome, and isolating a specific clone of the recombinant bacteriophage. In some embodiments, the selected wild-type bacteriophage is a Cronobacter-specific bacteriophage. In some embodiments, the selected wild-type bacteriophage is a myovirus (e.g., T4, T4-like, or Vil-like). In some embodiments, the selected wild-type bacteriophage infects Cronobacter spp. (e.g., bacteriophage Saka2 or Saka4). Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, likely myoviruses. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus or an Sp6-like virus). In other embodiments, the selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, likely a podovirus related to T7 phage.

[0011] In some embodiments, the step of preparing a homologous recombination plasmid / vector includes determining the native nucleotide sequence in the late region of the genome of the selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence comprising a codon-optimized indicator gene, and incorporating the sequence designed for homologous recombination into the plasmid / vector. The step of designing the sequence may include inserting a gene construct comprising a phage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene. In some embodiments, the phage late gene promoter is an exogenous promoter that is different from any endogenous promoter in the phage genome. Thus, in some methods, the homologous recombination plasmid comprises a bacteriophage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene.

[0012] Some embodiments of the present invention are compositions comprising the recombinant indicator bacteriophages described herein. For example, the compositions may comprise one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may comprise a cocktail of different indicator phages that may encode and express the same or different indicator proteins.

[0013] In some embodiments, the present invention encompasses a method for detecting a microorganism of interest in a sample, comprising incubating the sample with a recombinant bacteriophage that infects the microorganism of interest, wherein the recombinant bacteriophage contains an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the microorganism of interest in the sample.

[0014] In some embodiments of the method for preparing a recombinant indicator bacteriophage, the wild-type bacteriophage is a Cronobacter spp.-specific bacteriophage and the target pathogenic bacterium is a Cronobacter spp. In some embodiments, the wild-type bacteriophage specifically infects bacteria previously classified as Enterobacter sakazakii. In some embodiments, isolating specific clones of recombinant bacteriophage comprises a limiting dilution assay to isolate clones that exhibit expression of the indicator gene.

[0015] Another aspect of the present invention includes a method for detecting bacteria (e.g., Cronobacter spp.) in a sample, comprising incubating the sample with a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage, and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Cronobacter spp. is present in the sample. In some embodiments, the present invention includes a method for detecting Cronobacter spp. using a recombinant bacteriophage derived from a bacteriophage that targets Cronobacter spp. The sample may be a food sample or a water sample.

[0016] In some embodiments of methods for detecting bacteria, the sample is first incubated under conditions that favor growth for an enrichment period of 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, or 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, or 2 hours or less. In some embodiments, the sample is not enriched prior to detection. In some embodiments, the total time to result is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. In some embodiments, the signal to background ratio generated by detecting the indicator is at least 2.0, or at least 2.5, or at least 3.0. In some embodiments, the methods detect as few as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample of standard size for the food safety industry.

[0017] Further embodiments include systems and kits for detecting Cronobacter spp., wherein the system or kit comprises a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage. Some embodiments further comprise a substrate for reacting with an indicator to detect a soluble protein product expressed by the recombinant bacteriophage. These systems or kits may include features described with respect to the bacteriophages, compositions, and methods of the invention. In yet other embodiments, the invention includes a non-transitory computer-readable medium for use with a method or system according to the invention. [Brief explanation of the drawings]

[0018] The invention can be better understood with reference to the non-limiting drawings.

[0019] [Figure 1] 1 shows an indicator phage construct according to one embodiment of the present invention, illustrating the insertion of a gene construct comprising a luciferase gene, a bacteriophage late gene promoter, and a ribosome binding site (RBS) inserted into the late (class III) region of a bacteriophage. The promoter shown is upstream of an endogenous late gene (e.g., the gene for major capsid protein (MCP)) in addition to, or separate from, the endogenous late gene promoter.

[0020] [Figure 2]Figure 2 shows the genome of bacteriophage SAKA2 (a myovirus (related to T4 bacteriophage)) obtained from Cronobacter. A hypothetical gene homologous to the PG7 tail sheath protein surrounds the late gene region, which consists of structural genes encoding virion proteins. Other late genes shown are a known head vertex protein homolog, ORF58.1, likely a major capsid protein, and homologs of known outer head proteins. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided that a late gene promoter and / or other similar control elements are used.

[0021] [Figure 3] Figure 3 shows two homologous recombination plasmid construct designs for three different phages, each carrying a luciferase gene with approximately 500 bp of matching phage sequence upstream and downstream of the insertion site to facilitate homologous recombination. NANOLUC® luciferase is inserted into the pUC57.AmpR plasmid backbone with an upstream untranslated region containing a dedicated phage late gene promoter and ribosome entry site. Cronobacter phages Saka2 and Saka4 were newly isolated and sequenced phages, likely myoviruses. Each construct consisted of 500 bp of homologous sequence consisting of a fragment of the major capsid protein gene, followed by the T4 late gene promoter (which is in addition to the endogenous late gene promoter upstream of the major capsid protein in the phage genome), the luciferase gene, and approximately 500 bp of downstream matching sequence for homologous recombination. Saka9 and Saka10 are newly isolated and sequenced phages, likely podoviruses (related to the T7 phage), that require a T7-like late gene promoter instead of the T4 late gene promoter.

[0022] [Figure 4]Figure 4 shows the isolation of recombinant phage from the modification of bacteriophage using a plasmid construct (such as that shown in Figure 3) using a series of sequential infection and dilution steps to identify recombinant phage expressing an indicator gene.

[0023] [Figure 5] FIG. 5 illustrates the use of an indicator phage encoding a soluble luciferase to detect bacterial cells via detection of luciferase produced from replicating progeny phage during infection of the bacterial cells, in accordance with one embodiment of the present invention.

[0024] [Figure 6] FIG. 6 describes a filter plate assay for detecting bacteria of interest using modified bacteriophages according to one embodiment of the present invention, in which bacteria and recombinant phages are incubated on a filter plate and, after production of progeny bacteriophage, the indicator protein is detected directly without removal of the incubation medium.

[0025] [Figure 7] FIG. 7 shows a "No Concentration Assay" for detecting bacteria of interest using modified bacteriophages according to an embodiment of the present invention.

[0026] [Figure 8] FIG. 8 shows a hybrid immuno-phage (HIP) assay for detecting bacteria of interest using modified bacteriophages according to one embodiment of the present invention, which use antibodies against the microorganism to capture the microorganism on the surface of an assay well before incubation with a recombinant infectious agent carrying an indicator gene. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description of the Invention Disclosed herein are compositions, methods, and systems that exhibit surprising sensitivity for detecting microorganisms of interest (e.g., Cronobacter spp.) in test samples (e.g., biological, food, water, and environmental samples). Detection can be achieved in shorter time frames than previously thought possible using genetically modified infectious agents in assays performed without enrichment culture or, in some embodiments, with minimal incubation times (during which the microorganisms can potentially grow). Also surprising is the success of using potentially high multiplicities of infection (MOIs), or high concentrations of plaque-forming units (PFU), for incubation with test samples. Such high phage concentrations (PFU / mL) were previously claimed to be harmful in bacterial detection assays because they were claimed to cause "lysis from without." However, high concentrations of phage can facilitate the discovery, binding, and infection of small numbers of target cells.

[0028] The compositions, methods, systems, and kits of the invention may include infectious agents for use in detecting microorganisms such as Cronobacter spp. In certain embodiments, the invention may encompass compositions comprising a recombinant bacteriophage having an indicator gene inserted into the late gene region of the bacteriophage. In certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in the production of a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene (i.e., class III) region of the bacteriophage. The bacteriophage may be derived from a podovirus (e.g., T7, T7-like), a myovirus (e.g., T4, T4-like, ViI, ViI-like (or Vi1 virus, according to GenBank / NCBI)), a Cronobacter spp.-specific bacteriophage, or another wild-type or engineered bacteriophage. In some embodiments, the selected wild-type bacteriophage is Saka2 or Saka4. Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, likely myoviruses. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus) or an Sp6-like virus. In other embodiments, the selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, likely a podovirus related to T7 phage.

[0029] In some aspects, the present invention includes methods for detecting a microorganism of interest. The methods may use an infectious agent for the detection of the microorganism of interest (e.g., Cronobacter spp.). For example, in certain embodiments, the microorganism of interest is Cronobacter spp. and the infectious agent is Cronobacter spp. spp. Thus, in certain embodiments, the method may include detecting a bacterium of interest in a sample by incubating the sample with a recombinant bacteriophage that infects the bacterium of interest. In certain embodiments, the recombinant bacteriophage includes an indicator gene. The indicator gene may, in certain embodiments, be inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in production of an indicator protein product. The method may include detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample. In some embodiments, the indicator protein is soluble.

[0030] In certain embodiments, the present invention may include a system. The system may include at least some of the compositions of the present invention. The system may also include at least some of the components for performing the methods. In certain embodiments, the system is formulated as a kit. Thus, in certain embodiments, the present invention may include a system for rapid detection of a microorganism of interest, such as Cronobacter spp., in a sample, the system including components for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; and components for detecting the indicator moiety. In yet other embodiments, the present invention includes software for use with the methods or systems.

[0031] Thus, some embodiments of the present invention address this need by using a bacteriophage-based method to amplify a detectable signal indicating the presence of bacteria. In certain embodiments, as few as one bacterium can be detected. The principles applied herein can be applied to the detection of a variety of microorganisms. Due to the many binding sites for infectious agents on the surface of microorganisms, the ability to generate 100 or more progeny agents during infection, and the potential for high-level expression of the encoded indicator moiety, the infectious agent or indicator moiety may be more easily detectable than the microorganism itself. In this way, embodiments of the present invention can achieve very large signal amplification from even a single infected cell.

[0032] Aspects of the present invention utilize the high specificity of binding agents capable of binding to particular microorganisms, such as binding components of infectious agents, as a means of detecting and / or quantifying specific microorganisms in a sample. In some embodiments, the present invention utilizes the high specificity of infectious agents (e.g., bacteriophages).

[0033] In some embodiments, detection is achieved through an indicator moiety associated with a binding agent specific for the microorganism of interest. For example, an infectious agent may include an indicator moiety (e.g., a gene encoding a soluble indicator). In some embodiments, the indicator may be encoded by the infectious agent (e.g., a bacteriophage), and the bacteriophage is designated an indicator phage.

[0034] Some embodiments of the invention disclosed and described herein take advantage of the discovery that a single microorganism can bind a specific recognition factor (e.g., a phage). After infection and replication of the phage, the progeny phage can be detected via an indicator moiety expressed during phage replication. This principle allows for the amplification of an indicator signal from one or a few cells based on specific recognition of a microbial surface receptor. For example, by exposing a single bacterial cell to multiple phages and then allowing high-level expression of the encoded indicator gene product during phage amplification and replication, the indicator signal is amplified so that the single bacterium can be detected.

[0035] Embodiments of the methods and systems of the present invention can be applied to the detection and quantification of various microorganisms (e.g., bacteria) in various environments, including, but not limited to, the detection of pathogens from food, water, and commercial samples. The methods of the present invention provide rapid, high detection sensitivity and specificity. In some embodiments, detection is unexpectedly possible within a single replication cycle of the bacteriophage.

[0036] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein, as well as these techniques, are well known and commonly used in the art. Known methods and techniques are generally performed by conventional methods well known in the art and as described in the various general and more specific references discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with the laboratory procedures and techniques described herein is well known and commonly used in the art.

[0037] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0038] As used herein, the terms "a," "an," and "the" can refer to one or more unless otherwise noted.

[0039] Use of the term "or" is used to mean "and / or" unless expressly indicated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0040] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists from sample to sample.

[0041] The term "solid support" or "support" refers to a structure that provides a substrate and / or surface onto which a biomolecule can be attached. For example, a solid support can be an assay well (i.e., for example, a microtiter plate or a multiwell plate), or the solid support can be a filter, an array, or a location on a mobile support (e.g., beads) or membrane (e.g., a filter plate, latex particles, paramagnetic particles, or lateral flow strip).

[0042] The term "binding agent" refers to a molecule that can specifically and selectively bind to a second (i.e., different) molecule of interest. The interaction can be non-covalent, for example, as a result of hydrogen bonding, van der Waals interactions, or electrostatic or hydrophobic interactions, or the interaction can be covalent. The term "soluble binding agent" refers to a binding agent that is not associated (i.e., covalently or non-covalently bound) to a solid support.

[0043] As used herein, "analyte" refers to a molecule, compound, or cell being measured. An analyte of interest may, in certain embodiments, interact with a binding agent. As described herein, the term "analyte" may refer to a protein or peptide of interest. An analyte may be an agonist, antagonist, or modulator. Alternatively, an analyte may have no biological effect. Analytes may include small molecules, sugars, oligosaccharides, lipids, peptides, peptidomimetics, organic compounds, and the like.

[0044] The term "detectable moiety" or "detectable biomolecule" or "reporter" or "indicator" or "indicator moiety" refers to a molecule that can be measured in a quantitative assay. For example, an indicator moiety can include an enzyme that can be used to convert a substrate into a product that can be measured. An indicator moiety can be an enzyme (e.g., luciferase) that catalyzes a reaction that results in the emission of bioluminescence. Alternatively, an indicator moiety can be a radioisotope that can be quantified. Alternatively, an indicator moiety can be a fluorophore. Alternatively, other detectable molecules can be used.

[0045] As used herein, "bacteriophage" or "phage" includes one or more of several bacterial viruses. In this disclosure, the terms "bacteriophage" and "phage" refer to viruses that can invade living bacteria, fungi, mycoplasma, protozoa, yeast, and other microscopic living organisms and use them to replicate themselves, including viruses such as mycobacteriophage (e.g., for TB and paraTB), mycophage (e.g., for fungi), mycoplasma phage, and any other term. Here, "microscopic" means one millimeter or less in maximum dimension. Bacteriophages are viruses that naturally evolved to use bacteria as a means of replicating themselves. Phages do this by attaching themselves to bacteria, injecting their DNA (or RNA) into the bacteria, and inducing the bacteria to replicate the phage hundreds or even thousands of times. This is also called phage amplification.

[0046] As used herein, "late gene region" refers to a region of a viral genome that is transcribed late in the viral life cycle. The late gene region typically contains the most abundantly expressed genes (e.g., structural proteins assembled into the bacteriophage particle). Late genes are synonymous with class III genes and include genes with structural and assembly functions. For example, in phage T7, from 8 minutes post-infection until lysis, class I (e.g., RNA polymerase) is transcribed early, at 4-8 minutes, and class II at 6-15 minutes, with the timing of II and III genes overlapping. A late promoter is a promoter that is naturally located and active in such a late gene region.

[0047] As used herein, "enrichment culture" refers to traditional culture (e.g., incubation in a medium favorable for microbial growth) and should not be confused with other possible uses of the term "enrichment" (e.g., enrichment by removing a liquid component of a sample and concentrating the microorganisms contained therein) or other forms of enrichment that do not involve traditional promotion of microbial growth. Enrichment culture over a period of time may be used in some embodiments of the methods described herein.

[0048] As used herein, "recombinant" means a gene that is not found in any other way. "Modified" refers to genetic (i.e., nucleic acid) modification, usually done in a laboratory, to combine unmodified genetic material. This term is used interchangeably herein with the term "modified."

[0049] As used herein, "RLU" refers to the relative units of light emitted, as measured by a luminometer (e.g., GLOMAX® 96) or similar light-detecting instrument. For example, detection of the reaction between luciferase and an appropriate substrate (e.g., NANOLUC® and NANO-GLO®) is often reported in terms of the RLU detected.

[0050] As used herein, "time to results" refers to the total amount of time from the start of sample incubation until a result is generated. The time to results does not include any confirmatory testing time. Data collection can occur any time after a result is generated.

[0051] sample Each of the method and system embodiments of the present invention can allow for rapid detection and quantification of microorganisms in a sample. For example, methods according to the present invention can be performed in a shortened period of time with excellent results.

[0052] Bacterial cells that can be detected by the present invention include, but are not limited to, bacterial cells that are food- or water-borne pathogens.

[0053] The sample can be liquid, solid, or semi-solid. The sample can be a swab of a solid surface. Samples can include filters from environmental materials (e.g., water samples), or air or aerosol samples from cyclone collectors. The sample can be a sample of vegetables, meat, fish, poultry, peanut butter, processed foods, infant formula, milk powder, tea, starch, eggs, milk, cheese, or other dairy products.

[0054] In some embodiments, samples can be used directly in the detection methods of the present invention without preparation, concentration, or dilution. For example, liquid samples (including, but not limited to, milk and juice) can be assayed directly. Samples can be diluted or suspended in solutions, including, but not limited to, buffered solutions or bacterial culture media. Solid or semi-solid samples can be suspended in liquid by chopping, mixing, or macerating the solid in the liquid. Samples should be maintained within a pH range that promotes bacteriophage attachment to the host bacterial cells. Samples can also be diluted with divalent and monovalent cations (Na +, Mg 2+ , and Ca 2+ Preferably, the sample is maintained at a temperature that maintains the viability of any pathogen cells contained within the sample.

[0055] In some embodiments of the detection assay, the sample is maintained at a temperature that maintains the viability of any pathogen cells present in the sample. For example, during the step in which bacteriophage are attaching to bacterial cells, it is preferred to maintain the sample at a temperature that promotes bacteriophage attachment. During the step in which bacteriophage are replicating within infected bacterial cells or lysing such infected cells, it is preferred to maintain the sample at a temperature that promotes bacteriophage replication and lysis of the host. Such a temperature is at least about 25 degrees Celsius (C), more preferably about 45°C or less, and most preferably about 37°C.

[0056] The assay may include a variety of appropriate control samples, for example, a control sample containing no bacteriophage or a control sample containing bacteriophage without bacteria may be assayed as a control for background signal levels.

[0057] Indicator bacteriophage As described in more detail herein, the compositions, methods, systems, and kits of the present invention may include infectious agents for use in detecting pathogenic microorganisms. In certain embodiments, the present invention includes recombinant indicator bacteriophages, wherein the bacteriophage genome is genetically modified to include an indicator or reporter gene. In some embodiments, the present invention may include compositions comprising recombinant bacteriophages having an indicator gene integrated into the bacteriophage genome.

[0058] The recombinant indicator bacteriophage may contain a reporter or indicator gene. In certain embodiments of the infectious agent, the indicator gene does not encode a fusion protein. For example, in certain embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. In certain embodiments, the indicator gene may be inserted into the late gene region of the bacteriophage. Late genes encode structural proteins and are therefore generally expressed at higher levels than other phage genes. The late gene region may be a class III gene region and may include a gene for a major capsid protein.

[0059] Some embodiments include designing (and optionally preparing) a sequence for homologous recombination downstream of the major capsid protein gene. Other embodiments include designing (and optionally preparing) a sequence for homologous recombination upstream of the major capsid protein gene. In some embodiments, the sequence includes a codon-optimized reporter gene followed by an untranslated region. The untranslated region may include a phage late gene promoter and a ribosome entry site.

[0060] In some embodiments, the indicator bacteriophage is derived from T7, T4, or another similar phage. The indicator bacteriophage may also be derived from a T4-like, T7-like, ViI, ViI-like, Cronobacter spp.-specific bacteriophage, or another bacteriophage having a genome that shares at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology with a T4-like, T7-like, T4, T4-like, Cronobacter spp.-specific bacteriophage, ViI, or ViI-like (or Vi1 virus-like according to GenBank / NCBI) bacteriophage. In some embodiments, the selected wild-type bacteriophage is Saka2 or Saka4. Cronobacter spp. phages Saka2 and Saka4 are newly isolated and sequenced phages, likely myoviruses. In other embodiments, the selected wild-type bacteriophage is a podovirus (e.g., a T7-like virus) or an Sp6-like virus. In other embodiments, the selected wild-type bacteriophage is Saka10. Saka10 is a newly isolated and sequenced phage, likely a podovirus related to T7 phage. In some embodiments, the indicator phage is derived from a bacteriophage that is highly specific for a particular pathogenic microorganism. Genetic modification can avoid deletion of wild-type genes, so the modified phage can remain more similar to the wild-type infectious agent than many commercially available phages. Bacteriophages derived from the environment may be more specific to bacteria found in the environment and, thus, may be genetically distinct from commercially available phages.

[0061] Furthermore, phage genes considered non-essential may have unrecognized functions. For example, apparently non-essential genes may have important functions in increasing burst size, such as clever cleavage, adaptation, or trimming functions during assembly. Therefore, deleting genes and inserting indicators may be detrimental. Most phages can package DNA several percent larger than their native genome. In this regard, smaller indicator genes may be a more appropriate choice for modifying bacteriophages, especially those with smaller genomes. OpLuc and NANOLUC® proteins are only about 20 kDa (encoding about 500-600 bp), while FLuc is about 62 kDa (encoding about 1,700 bp). For comparison, the T7 genome is about 40 kbp, while the T4 genome is about 170 kbp and the genome of a Cronobacter-specific bacteriophage is about 157 kbp. Furthermore, the reporter gene should not be endogenously expressed by the bacteria (i.e., not be part of the bacterial genome), should generate a high signal-to-background ratio, and should be easily detectable in a timely manner. Promega's NANOLUC® is a modified Oplophorus gracilirostris (deep-sea shrimp) luciferase. In some embodiments, NANOLUC® in combination with Promega's NANO-GLO®, an imidazopyrazinone substrate (furimazine), can provide a robust signal with low background.

[0062] In some indicator phage embodiments, the indicator gene may be inserted into an untranslated region to avoid disrupting a functional gene, leaving the wild-type phage gene intact, which may result in greater fitness when infecting non-laboratory strains of bacteria. Additionally, including stop codons in all three reading frames may help increase expression by reducing read-through (also known as leaky expression). This strategy may also eliminate the possibility of low levels of fusion protein being made, which appears as a background signal (e.g., luciferase) that cannot be separated from the phage.

[0063] The indicator gene can express a variety of biomolecules. The indicator gene is a gene that expresses a detectable product or an enzyme that produces a detectable product. For example, in one embodiment, the indicator gene encodes a luciferase enzyme. Various types of luciferases can be used. In alternative embodiments, and as described in more detail herein, the luciferase is Oplophorus luciferase, firefly luciferase, Lucia luciferase, Renilla luciferase, or one of the engineered luciferases. In some embodiments, the luciferase gene is derived from Oplophorus. In some embodiments, the indicator gene is a genetically modified luciferase gene, such as NANOLUC®.

[0064] Thus, in some embodiments, the invention includes a genetically modified bacteriophage containing a non-bacteriophage indicator gene in the late (class III) gene region. In some embodiments, the non-native indicator gene is under the control of a late promoter. The use of a viral late gene promoter ensures that the reporter gene (e.g., luciferase) is not only expressed at high levels, like viral capsid proteins, but is also not silenced, like endogenous bacterial genes or even early viral genes.

[0065] In some embodiments, the late promoter is a T4-like, T7-like, or ViI-like promoter, or another phage promoter similar to that found in the selected wild-type phage (i.e., without genetic modification). The late gene region may be a Class III gene region, and the bacteriophage may be derived from a T7, T4, T4-like, ViI, ViI-like, Cronobacter spp.-specific bacteriophage, or another wild-type bacteriophage having a genome with at least 70%, 75%, 80%, 85%, 90%, or 95% homology to a T7, T4, T4-like, ViI, ViI-like, or Cronobacter-specific bacteriophage.

[0066] Genetic modifications to an infectious agent can include the insertion, deletion, or substitution of small nucleic acid fragments, substantial portions of genes, or entire genes. In some embodiments, the inserted or substituted nucleic acid comprises a non-native sequence. A non-native indicator gene can be inserted into the bacteriophage genome such that it is under the control of a bacteriophage promoter. Thus, in some embodiments, the non-native indicator gene is not part of a fusion protein. That is, in some embodiments, the genetic modification can be configured so that the indicator protein product does not contain polypeptides of the wild-type bacteriophage. In some embodiments, the indicator protein product is soluble. In some embodiments, the present invention encompasses methods for detecting bacteria of interest, comprising incubating a test sample with such recombinant bacteriophage.

[0067] In some embodiments, expression of the indicator gene in progeny bacteriophage after infection of a host bacterium results in a free, soluble protein product. In some embodiments, the non-native indicator gene is not linked to a gene encoding a phage structural protein and therefore does not result in a fusion protein. Unlike systems that use a fusion of a detection moiety to a capsid protein (i.e., a fusion protein), some embodiments of the present invention express a soluble indicator or reporter (e.g., a soluble luciferase). In some embodiments, the indicator or reporter ideally does not comprise the bacteriophage structure. That is, the indicator or reporter is not bound to the phage structure. Thus, the indicator or reporter gene is not fused to other genes in the recombinant phage genome. This can greatly increase the sensitivity of the assay (down to a single bacterium) and simplify the assay, allowing it to be completed in two hours or less for some embodiments, as opposed to the several hours required due to the additional purification steps required for constructs that generate detectable fusion proteins. Furthermore, fusion proteins may be less active than soluble proteins due to, for example, protein folding constraints that may alter the conformation of the enzyme active site or access to the substrate. For example, less than 2 hours may be sufficient for the assay if the concentration is 10 bacterial cells / mL sample.

[0068] Furthermore, fusion proteins by definition limit the number of moieties attached to a protein subunit on the bacteriophage. For example, using a commercially available system designed to serve as a platform for fusion proteins, approximately 415 copies of the fusion moiety are generated in each T7 bacteriophage particle, corresponding to approximately 415 copies of the gene 10B capsid protein. Without this constraint, infected bacteria would be expected to express more copies of the detection moiety (e.g., luciferase) than can fit on the bacteriophage. Furthermore, large fusion proteins (e.g., capsid-luciferase fusions) may inhibit assembly of the bacteriophage particle, thus resulting in fewer bacteriophage progeny. Therefore, a soluble, non-fused indicator gene product may be preferred.

[0069] In some embodiments, the indicator phage encodes a reporter, such as a detectable enzyme. The indicator gene product may produce light and / or be detectable by a color change. A variety of suitable enzymes are commercially available (e.g., alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc)). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0070] In some embodiments, the use of soluble detection moieties eliminates the need to remove contaminating parent phage from lysates of the infected sample cells. With a fusion protein system, any bacteriophage used to infect sample cells has an attached detection moiety and is indistinguishable from daughter bacteriophage that also contain the detection moiety. Because detection of sample bacteria relies on detection of newly created (de novo synthesized) detection moieties, the use of fusion constructs requires additional steps to separate the old (parent) moiety from the newly created (daughter bacteriophage) moiety. This can be accomplished by washing the infected cells multiple times prior to completion of the bacteriophage life cycle, inactivating excess parent phage after infection by physical or chemical means, and / or chemically modifying the parent bacteriophage with a binding moiety (e.g., biotin), which can then be bound and separated (e.g., by streptavidin-coated Sepharose beads). However, even with all these attempts at removal, parent phage can persist if a high concentration of parent phage is used to ensure infection of a small number of sample cells, creating a background signal that can obscure the detection of signal from progeny phage in infected cells.

[0071] In contrast, with the soluble detector moieties expressed in some embodiments of the invention, purification of the parent phage from the final lysate is not necessary because the parent phage does not have any detector moieties attached. Therefore, any detector moieties present after infection must be newly produced, indicating the presence of the infected bacterium or bacteria. To take advantage of this benefit, the generation and preparation of parent phage can include purifying the phage from any free detector moieties generated during parent bacteriophage generation in bacterial culture. Standard bacteriophage purification techniques can be used to purify some embodiments of phage according to the invention (e.g., sucrose density gradient centrifugation, cesium chloride isopycnic density gradient centrifugation, HPLC, size exclusion chromatography, etc.). chromatographies, and dialysis or derivative techniques (e.g., Amicon brand concentrators - Millipore, Inc.). Cesium chloride isopycnic ultracentrifugation can be used as part of the preparation of recombinant phage of the invention to separate parent phage particles from contaminating luciferase protein produced during propagation of the phage in the bacterial host. In this way, the parent recombinant bacteriophage of the invention are substantially free of any luciferase produced during production in the bacteria. Removal of residual luciferase present in the phage stock can substantially reduce the background signal observed when the recombinant bacteriophage is incubated with a test sample.

[0072] In some modified bacteriophage embodiments, the late promoter (a class III promoter, e.g., derived from T7, T4, ViI, or Saka) has high affinity for the RNA polymerase of the same bacteriophage, which transcribes the genes for the structural proteins assembled into bacteriophage particles. These proteins are the most abundant proteins made by the phage, because each bacteriophage particle contains tens or hundreds of copies of these molecules. Use of a viral late promoter can optimally ensure high-level expression of the luciferase detection moiety. Use of a late viral promoter derived from, specific for, or active under the original wild-type bacteriophage from which the indicator phage is derived (e.g., T4, T7, ViI, or Saka late promoter with a T4-based, T7-based, ViI-based, or Saka-based system) can further ensure optimal expression of the detection moiety. Use of standard bacterial (non-viral / non-bacteriophage) promoters can, in some cases, be detrimental to expression. Because these promoters are often downregulated during bacteriophage infection (as the bacteriophage prioritizes bacterial resources for phage protein production), in some embodiments, the phage is engineered to encode and express high levels of a soluble (free) indicator moiety, preferably using a location in the genome that does not limit expression to the number of subunits of the phage structural components.

[0073] Compositions of the invention may include one or more wild-type or genetically modified infectious agents (e.g., bacteriophages) and one or more indicator genes. In some embodiments, the compositions may include a cocktail of different indicator phages that may encode and express the same or different indicator proteins. In some embodiments, the cocktail of bacteriophages includes at least two different types of recombinant bacteriophages.

[0074] Method for preparing indicator bacteriophages An embodiment of the method for generating indicator bacteriophages begins with the selection of a wild-type bacteriophage for genetic modification. Some bacteriophages are highly specific for target bacteria. This provides an opportunity for highly specific detection.

[0075] Thus, the methods of the present invention take advantage of the high specificity of binding agents associated with infectious agents that recognize and bind to specific microorganisms of interest as a means of amplifying the signal and thereby detecting low levels of microorganisms (e.g., single microorganisms) present in a sample. For example, infectious agents (e.g., bacteriophages) specifically recognize surface receptors on specific microorganisms and therefore specifically infect those microorganisms. Thus, these infectious agents can be suitable binding agents for targeting microorganisms of interest.

[0076] Some embodiments of the present invention utilize the binding specificity and high-level gene expression capabilities of recombinant bacteriophage for rapid and sensitive targeting to infect and facilitate detection of bacteria of interest. In some embodiments, the Cronobacter-specific bacteriophage is genetically modified to contain a reporter gene. In some embodiments, the late gene region of the bacteriophage is genetically modified to contain the reporter gene. In some embodiments, the reporter gene is located downstream of the major capsid gene. In other embodiments, the reporter gene is located upstream of the major capsid gene. In some embodiments, the inserted genetic construct further comprises a dedicated exogenous promoter to drive expression of the indicator gene. The exogenous promoter is in addition to any endogenous promoters in the phage genome. Because bacteriophages generate polycistronic mRNA transcripts, only a single promoter is required upstream of the first gene / cistron in the transcript. Conventional recombinant constructs simply use the endogenous bacteriophage promoter to drive the inserted gene. In contrast, adding an additional promoter upstream of the reporter gene and ribosome binding site can increase gene expression by acting as a second initiation site for transcription. The complex and compact genomes of viruses often contain overlapping genes in different frames, sometimes in two different orientations.

[0077] Some embodiments of methods for preparing recombinant indicator bacteriophage include the steps of selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, such as Cronobacter spp., preparing a homologous recombination plasmid / vector containing an indicator gene, transforming the homologous recombination plasmid / vector into the target pathogenic bacterium, infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby causing homologous recombination between the plasmid / vector and the bacteriophage genome, and isolating a specific clone of the recombinant bacteriophage.

[0078] Various methods for designing and preparing homologous recombination plasmids are known. Various methods for transforming bacteria with plasmids are known, including heat shock, F-pilus-mediated bacterial conjugation, electroporation, and other methods. Various methods for isolating specific clones after homologous recombination are also known. Some method embodiments described herein utilize specific strategies.

[0079] Thus, some embodiments of methods for preparing indicator bacteriophages include selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium, determining the native sequence in the late region of the genome of the selected bacteriophage, annotating the genome and identifying the major capsid protein gene of the selected bacteriophage, designing sequences for homologous recombination adjacent to the major capsid protein gene, wherein the sequences comprise a codon-optimized reporter gene, incorporating the sequences designed for homologous recombination into a plasmid / vector, transforming the plasmid / vector into the target pathogenic bacterium, selecting for the transformed bacteria, infecting the transformed bacteria with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid and the bacteriophage genome, determining the titer of the resulting recombinant bacteriophage lysate, and performing a limiting dilution assay to enrich for and isolate the recombinant bacteriophage. Some embodiments further include repeating the limiting dilution and titering steps as necessary after the first limiting dilution assay until recombinant bacteriophage represent a detectable proportion of the mixture. For example, in some embodiments, the limiting dilution and titering steps can be repeated until at least 1 / 30 of the bacteriophage in the mixture are recombinant before isolating specific clones of recombinant bacteriophage. A 1:30 recombinant:wild-type ratio is expected in some embodiments to yield an average of 3.2 transducing units (TU) per 96 plaques (e.g., in a 96-well plate). The initial ratio of recombinant phage:wild-type phage can be determined by performing a limiting dilution assay based on TCID50 (tissue culture infectious dose 50%), as previously described in U.S. Patent Application No. 15 / 409,258. By Poisson distribution, a 1:30 ratio results in a 96% chance of observing at least 1 TU in any of the 96 wells.

[0080] FIG. 1 shows a schematic representation of the genomic structure of a recombinant indicator bacteriophage of the present invention. For the embodiment shown in FIG. 1, the detection moiety is encoded by a luciferase gene 100 inserted within a late (class III) gene 110, which is expressed late in the viral life cycle. Late genes are generally expressed at higher levels than other phage genes because they encode structural proteins. Thus, in the recombinant phage embodiment shown in FIG. 1, the indicator gene (i.e., luciferase) is inserted into the late gene region, immediately following the major capsid protein (MCP) gene 120, a construct containing the luciferase gene 100. In some embodiments, the construct shown in FIG. 1 may contain stop codons in all three reading frames to ensure that luciferase is not incorporated into the MCP gene product via the creation of a fusion protein. As also shown in FIG. 1, the construct may contain an additional, dedicated late promoter 130 to drive transcription and expression of the luciferase gene. The construct also contains a ribosome binding site (RBS) 140. This construct ensures that soluble luciferase is produced, such that expression is restricted to the number of unique capsid proteins in the phage display system.

[0081] As noted herein, in certain embodiments, it may be preferable to utilize infectious agents that have been isolated from the environment for the production of the infectious agents of the present invention. In this manner, naturally occurring, microorganism-specific infectious agents can be produced.

[0082] For example, Figure 2 shows the genome of bacteriophage SAKA2 (a wild-type bacteriophage that specifically infects Cronobacter spp.). As discussed in the Examples, the major capsid protein and various other structural genes are located within the late gene region consisting of structural genes encoding virion proteins. A hypothetical gene homologous to the PG7 tail sheath protein gene (nucleotides 74424-77221) is located adjacent to the late gene region consisting of structural genes encoding virion proteins. Other suggested late genes are a homolog of the known head protein (nucleotides 77115-77653), ORF58.1 (nucleotides 77693-78610), a likely major capsid protein, and a homolog of the known outer head protein (nucleotides 80248-81840). Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided the late gene promoter and / or other similar regulatory elements are used.

[0083] Numerous known methods and commercial products are available for preparing plasmids. For example, PCR, site-directed mutagenesis, restriction digestion, ligation, cloning, and other techniques can be used in combination to prepare plasmids. Synthetic plasmids can also be ordered commercially (e.g., GeneWiz). Cosmids can also be used to selectively edit bacteriophage genomes, or the CRISPR / CAS9 system can be used. Some embodiments of methods for preparing recombinant indicator bacteriophages include designing a plasmid that can readily recombine with a wild-type bacteriophage genome to generate a recombinant genome. In designing the plasmid, some embodiments include adding a codon-optimized reporter gene, such as a luciferase gene. Some embodiments further include adding elements to an upstream untranslated region. For example, in designing a plasmid for recombination with a Cronobacter-specific bacteriophage genome, an upstream untranslated region can be added between the sequence encoding the C-terminus of the gp23 / major capsid protein and the start codon of the NANOLUC® reporter gene. The untranslated region may contain a promoter, such as a T4, T4-like, T7, T7-like, Cronobacter-specific bacteriophage, ViI, or ViI-like promoter. The untranslated region may also contain a ribosome entry / binding site (RBS), also known as a "Shine-Dalgarno sequence" in bacterial systems. Either or both of these elements, or other untranslated elements, may be embedded within a short upstream untranslated region made of random sequence containing approximately the same GC content as the rest of the phage genome. The random region should not contain an ATG sequence, since this acts as the start codon.

[0084] Compositions of the invention can include various infectious agents and / or indicator genes. For example, Figure 3 shows two homologous recombination plasmid constructs used in generating indicator phage specific for Cronobacter spp. Constructs were made and used in recombination with Cronobacter spp. phage Saka2, Cronobacter spp. phage Saka4, or Cronobacter spp. phage Saka10 to generate recombinant bacteriophages of the invention. The first construct in Figure 3 shows a general schematic of the recombinant plasmids used for homologous recombination insertion of NANOLUC® luciferase into both Cronobacter spp. phages Saka2 and Saka4, each with 500 bp of upstream and downstream homologous sequence corresponding to the respective phage:homologous recombination plasmids pUC57.HR.Saka2.NANOLUC® and pUC57.HR.Saka4.NANOLUC®.

[0085] The bottom construct in Figure 3 shows the recombinant plasmid used for homologous recombination insertion of NANOLUC® luciferase into Saka10 (podovirus) to target Cronobacter strains that are poorly infected by either Saka2 or Saka4: homologous recombination plasmid pUC57.HR.Saka10.NANOLUC®.

[0086] In certain embodiments, the plasmid is designated pUC57.HR.Saka2.NanoLuc. The detection / indicator portion is encoded by the NANOLUC® reporter gene 300. The insert (represented by a series of boxes) is in the standard AmpR version 310 of pUC57. The upstream homologous recombination region consists of 500 bp of the major capsid protein C-terminal fragment 320, a T4-like phage late promoter consensus sequence in the 5' untranslated region, and a Shine-Dalgarno ribosome entry / binding site 330. The codon-optimized NANOLUC® reporter gene 300 immediately follows. A downstream untranslated region (UTR) consisting of the homologous recombination 340 and hypothetical protein N-terminal fragment is at the end of the homologous recombination region.

[0087] In certain embodiments, the plasmid is designated pUC57.HR.Saka10.NanoLuc. The detection / indicator portion is encoded by the NanoLuc® reporter gene 380. The insert (represented by a series of boxes) is in the standard AmpR version 360 of pUC57. The upstream homologous recombination region consists of 500 bp of the major capsid protein C-terminal fragment 360, a T7 phage late promoter consensus sequence in the 5' untranslated region, and a Shine-Dalgarno ribosome entry / binding site 370. The codon-optimized NANOLUC® reporter gene 350 immediately follows. A downstream untranslated region (UTR) consisting of the homologous recombination 380 and hypothetical protein N-terminal fragment is at the end of the homologous recombination region. The major capsid protein fragment is part of a structural gene encoding a virion protein. Because these virion proteins are expressed at very high levels, any gene inserted into this region can be expected to have similar expression levels, provided that a late gene promoter and / or other similar control elements are used.

[0088] In some embodiments, indicator phage according to the present invention comprises a Cronobacter-specific bacteriophage genetically engineered to contain a reporter gene (e.g., a luciferase gene). For example, the indicator phage can be a Cronobacter spp.-specific bacteriophage whose genome contains the sequence of the NANOLUC® gene. The recombinant Cronobacter-specific NanoLuc bacteriophage genome can further comprise a T4, T7, Cronobacter-specific, ViI, Saka bacteriophage consensus promoter, or another late promoter. In a further embodiment, the promoter is an exogenous promoter. Insertion of an exogenous promoter to drive expression of the indicator gene is advantageous in that expression is limited by the expression of other phage proteins (e.g., the major capsid protein).

[0089] Thus, in an embodiment of the recombinant phage produced as a result of recombination, the indicator gene (i.e., NANOLUC®) is inserted into the late gene region immediately downstream of the gene encoding the major capsid protein, thus creating a recombinant bacteriophage genome containing the NANOLUC® gene. The construct further includes a T4, T7, Cronobacter-specific bacteriophage, ViI consensus promoter, or another late promoter or another suitable promoter to drive transcription and expression of the luciferase gene. The construct may also include a composite untranslated region synthesized from several UTRs. This construct ensures that a soluble luciferase is produced, such that expression is not limited by the number of unique capsid proteins in the phage display system.

[0090] Figure 4 shows the isolation of recombinant phage from a mixture of wild-type and recombinant bacteriophage resulting from homologous recombination. In a first step 402, Cronobacter spp. bacteria transformed with a homologous recombination plasmid are infected with Cronobacter spp. bacteriophage Saka2, resulting in progeny phage 434 having a mixture of parental and recombinant phage with a very low ratio of wild-type to recombinant phage. The resulting recombinant phage mixture is diluted 404 into 96-well plates 406 to give an average of 5 recombinant transducing units (TU) / plate (9.3 PFU / well). The 96-well plates are assayed for luciferase activity to identify wells 436 containing recombinant phage compared to wells 440 containing wild-type bacteriophage. Bacteria 438 are added 408; for example, each well may contain approximately 50 μL of turbid Cronobacter culture. This allows the phage to replicate and produce luciferase enzyme 442. After a 5-hour incubation at 37°C, as shown in 410, wells can be screened for the presence of luciferase 442. Any positive wells will likely have been inoculated with a single recombinant phage, and at this stage the mixture may contain a ratio of approximately 10 wild-type phage: 1 recombinant (enrichment over the original ratio). If necessary (i.e., if the recombinant:total ratio is lower than 1:30), progeny from this enriched culture 412 can be subjected to a further limiting dilution assay 414 to increase the ratio and determine the actual concentration of recombinant phage transducing units. For example, if the ratio was 1:384 recombinant:PFU, then 1920 total contaminating phage (5 x 384 = 1920) per 96-well plate 416 along with approximately 5 recombinant TU may be aliquoted 414 from the previous positive wells, resulting in an approximate inoculation of 20 mostly wild-type phage / well (1920 PFU / 96 well = 20 PFU / well) 420 of the second dilution assay plate. Any positive luciferase wells will likely be inoculated with a single recombinant along with 19 wild-type phage. These wells can be analyzed for the presence of luciferase 442.

[0091] After addition of bacteria and incubation (e.g., 5 hours at 37°C) 418, soluble luciferase and phage are present in approximately 20 total:1 recombinants 420. This ratio can be verified by TU50 titration of the recombinants, a limiting dilution assay based on a tissue culture infectious dose 50 (TCID50) assay that scores luciferase activity instead of cell killing, and a plaque assay for total PFU. Finally, a plaque assay can be performed 422 to screen for recombinants expressing luciferase 446. A small number of individual (e.g., n=48) plaques may be individually picked and screened in a third multiwell plate 426 for luciferase activity 436. In one embodiment, this approach should ensure that enough plaques are screened so that approximately 3 recombinants are present in the mixture of plaques being screened based on the known ratio of recombinants to total phage. One plaque can be picked from the plate into each well of a 96-well plate 424, and a luciferase assay can be performed 426 to determine which wells contained phage exhibiting luciferase activity 442. Wells exhibiting luciferase activity 428 represent pure recombinant phage 434, while wells without luciferase activity 430 represent pure wild-type phage 432.

[0092] Individual plaques can then be suspended in buffer (e.g., 100 μL TMS) or medium, and aliquots (e.g., about 5 μL) can be added to wells containing turbid Cronobacter cultures and assayed after incubation (e.g., about 45 minutes to 1 hour at 37°C). Positive wells are predicted to contain pure cultures of recombinant phage. Certain embodiments may include additional rounds of plaque purification.

[0093] Thus, as illustrated by Figure 4, recombinant phage generated by homologous recombination of a plasmid designed for recombination with a wild-type phage genome can be isolated from a mixture containing a very small percentage (e.g., 0.005%) of the total phage genome. After isolation, large-scale production can be performed to obtain high-titer recombinant indicator phage stocks suitable for use in Cronobacter spp. detection assays. Furthermore, cesium chloride isopycnic density gradient centrifugation can be used to separate phage particles from contaminating luciferase protein to reduce background.

[0094] Methods using infectious agents to detect Cronobacter spp. As noted herein, in certain embodiments, the present invention may include methods for using infectious particles to detect microorganisms. The methods of the present invention may be embodied in a variety of ways.

[0095] In one embodiment, the invention may include a method for detecting a bacterium of interest in a sample, comprising incubating the sample with a bacteriophage that infects the bacterium of interest, wherein the bacteriophage contains an indicator gene, such that expression of the indicator gene during bacteriophage replication after infection of the bacterium of interest results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates that the bacterium of interest is present in the sample.

[0096] In certain embodiments, assays can be performed utilizing a general concept that can be modified to accommodate different sample types or sizes and assay formats. Embodiments using recombinant bacteriophages of the invention (i.e., indicator bacteriophages) can be performed in the following time periods, depending on the sample type, sample size, and assay format: 1.5 hours, 2.0 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4.0 hours, 4.5 hours, 5.0 hours, 5.5 hours, 6.0 hours, 6.5 hours, 7.0 hours, 7.5 hours, 8.0 hours, 8.5 hours, 9.0 hours, 9.5 hours, 10.0 hours, 10.5 hours, 11.0 hours, 11.5 hours, 12 hours, 14 hours, 16 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, , 12.5 hours, 13.0 hours, 13.5 hours, 14.0 hours, 14.5 hours, 15.0 hours, 15.5 hours, 16.0 hours, 16.5 hours, 17.0 hours, 17.5 hours, 18.0 hours, 18.5 hours, 19.0 hours, 19.5 hours, 20.0 hours, 21.0 hours, 21.5 hours, 22.0 hours, 22.5 hours, 23.0 hours, 23.5 hours, 24.0 hours, 24.5 hours, 25.0 hours, 25.5 hours, or 26.0 hours, may enable rapid detection of specific bacterial strains such as Cronobacter spp. For example, the amount of time required may be slightly shorter or longer depending on the strain of bacteriophage and the strain of bacteria being detected in the assay, the type and size of the sample being tested, the conditions required for target viability, the complexity of the physical / chemical environment, and the concentration of "endogenous" non-target contaminating bacteria.

[0097] Figure 5 shows a strategy for using indicator phage to produce soluble luciferase according to an embodiment of the present invention. In this method, phage (e.g., T7, T4, Saka2, Saka4, Saka9, or Saka10 phage) can be engineered to express soluble luciferase during phage replication. Luciferase expression is driven by a viral capsid promoter (e.g., the late promoter of bacteriophage T7 or T4), resulting in high expression. Because parent phage do not contain luciferase, the luciferase detected in the assay must arise from replicating progeny phage during infection of the bacterial cells. Therefore, it is generally not necessary to separate the parent phage from the progeny phage.

[0098] In these experiments, at least a portion of a sample 500 containing bacteria 502 to be quantified is placed in a spin column filter, centrifuged to remove the LB broth, and an appropriate multiplicity of phage 504 genetically engineered to express soluble luciferase 503 is added. The infected cells can be incubated for a sufficient time (e.g., 30-120 minutes at 37°C) for progeny phage replication and cell lysis to occur. The parent phage 504 and progeny phage 516 + free luciferase 503 in the lysate can then be collected, for example, by centrifugation, and the level of luciferase in the filtrate can be quantified using a luminometer 518. Alternatively, a high-throughput method can be used in which bacterial samples are applied to a 96-well filter plate, and after all of the above-listed procedures have been performed, the original 96-well filter plate can be directly assayed for luciferase without the final centrifugation step.

[0099] Figure 6 illustrates a filter plate assay for detecting bacteria of interest using engineered bacteriophage according to one embodiment of the present invention. Briefly, a sample 616 containing bacteria of interest 618 is added to a well 602 of a multi-well filter plate 604 and concentrated by centrifugation 606 to remove liquid from the sample. Genetically engineered phage 620 is added to the well and incubated with additional media for a period of time sufficient for adsorption 608, followed by infection of the target bacteria and the phage life cycle 610 (e.g., approximately 45 minutes). Finally, a luciferase substrate is added and reacts with any luciferase present 624. The resulting luminescence is measured 614 in a luminometer that detects luciferase activity 626.

[0100] In certain embodiments, the assay can be performed without concentrating bacteria on or near a capture surface. Figure 7 illustrates a "No Concentration Assay" for detecting bacteria of interest using modified bacteriophage according to one embodiment of the present invention. Aliquots of indicator phage 714 are dispensed into individual wells 702 of a multiwell plate 704, followed by the addition of a test sample aliquot containing bacteria 712 and incubation 706 (e.g., 45 minutes at 37°C) for a period sufficient for the phage to replicate and produce a soluble indicator 716 (e.g., luciferase). The plate wells 708 containing soluble indicator and phage can then be assayed 710 to measure the indicator activity 718 on the plate (e.g., luciferase assay). In this embodiment, the test sample is not concentrated (e.g., by centrifugation) but simply incubated directly with indicator phage for a period of time and then assayed for luciferase activity.

[0101] In some embodiments, the sample can be enriched prior to testing by incubation in conditions conducive to growth. In such embodiments, the enrichment period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours or longer, depending on the type and size of the sample.

[0102] In some embodiments, the indicator bacteriophage comprises a detectable indicator moiety, and infection of a single pathogenic cell (e.g., a bacterium) can be detected by an amplified signal generated via the indicator moiety. Thus, the method can include detecting the indicator moiety generated during phage replication, where detection of the indicator indicates that the bacterium of interest is present in the sample.

[0103] In one embodiment, the present invention may comprise a method for detecting a bacterium of interest in a sample, comprising incubating the sample with a recombinant bacteriophage that infects the bacterium of interest, wherein the recombinant bacteriophage comprises an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product, and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of the bacterium of interest in the sample. In some embodiments, the amount of indicator moiety detected corresponds to the amount of the bacterium of interest present in the sample.

[0104] As described in more detail herein, the methods and systems of the present invention can utilize a range of concentrations of parent indicator bacteriophage to infect bacteria present in a sample. In some embodiments, the indicator bacteriophage is added to the sample at a concentration sufficient to rapidly find, bind to, and infect target bacteria present in the sample in very low numbers, such as single cells. In some embodiments, the phage concentration can be sufficient to find, bind to, and infect target bacteria in less than one hour. In other embodiments, these events can occur in less than two hours, or less than three hours, after adding the indicator phage to the sample. For example, in certain embodiments, the bacteriophage concentration for the incubating step is 1 x 10 5 PFU / mL or higher than 1 × 10 6 PFU / mL or higher than 1 × 10 7 Higher than PFU / mL.

[0105] In certain embodiments, the recombinant infectious agent can be purified to be free of any residual indicator protein that may be produced during production of the infectious agent stock. Thus, in certain embodiments, the recombinant bacteriophage can be purified using cesium chloride isopycnic density gradient centrifugation prior to incubation with the sample. When the infectious agent is a bacteriophage, this purification can have the added benefit of removing bacteriophage that do not have DNA (i.e., empty phage or "ghosts").

[0106] In some embodiments of the methods of the present invention, the microorganisms can be detected without any isolation or purification of the microorganisms from the sample. For example, in certain embodiments, a sample containing one or several target microorganisms can be applied directly to an assay vessel (e.g., a spin column, a microtiter well, or a filter), and the assay is performed in the assay vessel. Various embodiments of such assays are disclosed herein.

[0107] Test sample aliquots can be dispensed directly into wells of a multiwell plate, indicator phage can be added, and after a sufficient period for infection, lysis buffer can be added as well as a substrate for the indicator moiety (e.g., luciferase substrate for a luciferase indicator) and assayed for detection of the indicator signal. Some embodiments of the method can be performed on a filter plate. Some embodiments of the method can be performed with or without concentration of the sample prior to infection with indicator phage.

[0108] For example, in many embodiments, multi-well plates are used to perform the assay. The choice of plate (or any other container in which the detecting step can be performed) can affect the detecting step. For example, some plates may contain a colored or white background, which can affect the detection of light emission. Generally, white plates have higher sensitivity but also produce higher background signals. Other colors of plates may produce lower background signals but may have slightly lower sensitivity. Furthermore, one reason for background signal is light leakage from one well to another adjacent well. Some plates have white wells, while the rest of the plate is black. This allows for a high signal within the well but prevents light leakage from well to well, thus reducing background. Thus, the choice of plate or other assay container can affect the sensitivity and background signal for the assay.

[0109] The methods of the invention may include various other steps to increase sensitivity. For example, as discussed in more detail herein, the methods may include washing the captured and infected bacteria after adding the bacteriophage but before incubation to remove excess parent bacteriophage and / or luciferase or other reporter proteins that contaminate the bacteriophage preparation.

[0110] In some embodiments, detection of the target microorganism can be completed without the need to culture the sample as a method of increasing the population of the microorganism. For example, in certain embodiments, the total time required for detection is less than 26.0, 25.0, 24.0, 23.0, 22.0, 21.0, 20.0, 19.0, 18.0, 17.0, 16.0, 15.0, 14.0, 13.0, 12.0, 11.0, 10.0, 9.0, 8.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.5, 2.0, 1.5, 1.0, 45, or 30 minutes. Minimizing time to results is critical in food and environmental testing for pathogens.

[0111] In contrast to assays known in the art, the methods of the present invention can detect individual microorganisms. Thus, in certain embodiments, the methods can detect ≦10 cells of the microorganism (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 microorganisms) present in a sample. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Cronobacter spp. In one embodiment, the recombinant bacteriophage can distinguish Cronobacter spp. in the presence of other types of bacteria. In certain embodiments, the recombinant bacteriophage can be used to detect a single bacterium of a specific type in a sample. In certain embodiments, the recombinant bacteriophage detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in a sample.

[0112] Thus, aspects of the present invention provide methods for detecting a microorganism in a test sample via an indicator moiety. In some embodiments, when the microorganism of interest is a bacterium, the indicator moiety can be associated with an infectious agent (e.g., an indicator bacteriophage). The indicator moiety can react with a substrate to emit a detectable signal or can emit an intrinsic signal (e.g., a fluorescent protein). In some embodiments, the detection sensitivity can reveal the presence of as few as 50, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 cells of the microorganism of interest in a test sample. In some embodiments, as few as one cell of the microorganism of interest can produce a detectable signal. In some embodiments, the bacteriophage is a T4-like or ViI-like bacteriophage. In some embodiments, the recombinant bacteriophage is derived from a Cronobacter-specific bacteriophage. In certain embodiments, the recombinant Cronobacter-specific bacteriophage is highly specific for Cronobacter spp.

[0113] In some embodiments, the indicator moiety encoded by the infectious agent may be detectable during or after replication of the infectious agent. Many different types of detectable biomolecules suitable for use as indicator moieties are known in the art, and many are commercially available. In some embodiments, the indicator phage contains an enzyme, which serves as the indicator moiety. In some embodiments, the genome of the indicator phage is modified to encode a soluble protein. In some embodiments, the indicator phage encodes a detectable enzyme. The indicator may emit light and / or be detectable by a color change in a converted substrate. Various suitable enzymes are commercially available, such as alkaline phosphatase (AP), horseradish peroxidase (HRP), or luciferase (Luc). In some embodiments, these enzymes may serve as the indicator moiety. In some embodiments, firefly luciferase is the indicator moiety. In some embodiments, Oplophorus luciferase is the indicator moiety. In some embodiments, NANOLUC® is the indicator moiety. Other engineered luciferases or other enzymes that generate a detectable signal may also be suitable indicator moieties.

[0114] Thus, in some embodiments, the recombinant bacteriophage of the present methods, systems, or kits is prepared from a wild-type Cronobacter-specific bacteriophage. In some embodiments, the indicator gene encodes a protein that emits an intrinsic signal, such as a fluorescent protein (e.g., green fluorescent protein). The indicator may emit light and / or be detectable by a color change. In some embodiments, the indicator gene encodes an enzyme (e.g., luciferase) that interacts with a substrate to generate a signal. In some embodiments, the indicator gene is a luciferase gene. In some embodiments, the luciferase gene is one of Oplophorus luciferase, firefly luciferase, Renilla luciferase, External Gaussia luciferase, Lucia luciferase, or an engineered luciferase, such as NANOLUC®, Rluc8.6-535, or orange nano-lantern.

[0115] Detecting the indicator may include detecting light emission. In some embodiments, a luminometer may be used to detect the reaction of an indicator (e.g., luciferase) with a substrate. Detection of RLUs may be achieved with a luminometer, or other machines or devices may also be used. For example, a spectrophotometer, CCD camera, or CMOS camera may detect color changes and other light emissions. While absolute RLUs are important for detection, a high signal-to-background ratio (e.g., >2.0, >2.5, or >3.0) is also required to reliably detect single cells or small numbers of cells.

[0116] In some embodiments, the indicator phage is genetically engineered to contain a gene for an enzyme (e.g., luciferase) that is produced only upon infection of the bacterium that the phage specifically recognizes and infects. In some embodiments, the indicator moiety is expressed late in the viral life cycle. In some embodiments, as described herein, the indicator is a soluble protein (e.g., soluble luciferase) and is not fused to a phage structural protein that limits its copy number.

[0117] Thus, in some embodiments utilizing indicator phage, the present invention includes a method for detecting a microorganism of interest, comprising capturing at least one sample bacterium; incubating the at least one bacterium with a plurality of indicator phage; allowing time for infection and replication to produce progeny phage and express a soluble indicator moiety; and detecting the progeny phage, preferably the indicator, wherein detection of the indicator indicates the presence of the bacterium in the sample.

[0118] For example, in some embodiments, test sample bacteria may be captured by binding to the surface of a plate or by filtering the sample through a bacteriological filter (e.g., a 0.45 μm pore size spin filter or plate filter). In one embodiment, the infectious agent (e.g., indicator phage) is added in a minimal volume to the sample captured directly on the filter. In one embodiment, the microorganisms captured on the filter or plate surface are then washed one or more times to remove excess unbound infectious agent. In one embodiment, medium (e.g., Luria-Bertani broth (also referred to herein as LB), buffered peptone water (also referred to herein as BPW), or tryptic or tryptone soy broth (also referred to herein as TSB)) may be added for an additional incubation period to allow bacterial cells and phage replication and high-level expression of the gene encoding the indicator moiety. However, a surprising aspect of some embodiments of the test assay is that the incubation step with the indicator phage need only be long enough for a single phage life cycle. The amplifying power of using bacteriophage was previously thought to require more time, as the phage replicates over several cycles. A single replication cycle of the indicator phage can be sufficient to facilitate sensitive and rapid detection in accordance with some embodiments of the present invention.

[0119] In some embodiments, an aliquot of a test sample containing bacteria may be applied to a spin column, and after infection with recombinant bacteriophage and optional washing to remove any excess bacteriophage, the amount of soluble indicator detected is proportional to the amount of bacteriophage produced by the infected bacteria.

[0120] Soluble indicators (e.g., luciferase) released into the surrounding liquid upon lysis of the bacteria can then be measured and quantified. In one embodiment, the solution is centrifuged through a filter and the filtrate is collected in a new container for assay (e.g., in a luminometer) after which a substrate for the indicator enzyme (e.g., luciferase substrate) is added. Alternatively, the indicator signal can be measured directly on the filter.

[0121] In various embodiments, the purified parent indicator phage does not contain the detectable indicator itself, because the parent phage can be purified before being used to incubate with a test sample. Expression of late (class III) genes occurs late in the viral life cycle. In some embodiments of the invention, the parent phage can be purified to eliminate any present indicator protein (e.g., luciferase). In some embodiments, expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product. Thus, in many embodiments, it is not necessary to separate the parent phage from the progeny phage prior to the detection step. In one embodiment, the microorganism is a bacterium and the indicator phage is a bacteriophage. In one embodiment, the indicator moiety is a soluble luciferase, which is released upon lysis of the host microorganism.

[0122] Thus, in an alternative embodiment, the indicator substrate (e.g., luciferase substrate) can be incubated with the portion of the sample that remains on the filter or that remains bound to the plate surface. Thus, in some embodiments, the solid support is a 96-well filter plate (or a regular 96-well plate), and the substrate reaction can be detected by placing the plate directly into the luminometer.

[0123] For example, in one embodiment, the invention may include a method for detecting Cronobacter spp., comprising the steps of infecting cells captured on a 96-well filter plate with multiple parent indicator phages capable of expressing luciferase upon infection; washing away excess phages; adding LB broth and allowing time for phages to replicate and lyse the specific Cronobacter spp. targets (e.g., 30-120 minutes); and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity indicates the presence of the Cronobacter spp. in the sample.

[0124] In another embodiment, the present invention may include a method for detecting Cronobacter spp., comprising the steps of infecting cells in a liquid solution or suspension in a 96-well plate with multiple parent indicator phages capable of expressing luciferase upon infection; allowing time (e.g., 30 to 120 minutes) for the phage to replicate and lyse the specific Cronobacter spp. target; and detecting the indicator luciferase by adding a luciferase substrate and measuring luciferase activity directly in the 96-well plate, wherein detection of luciferase activity indicates the presence of the Cronobacter spp. in the sample. In such embodiments, a capture step is not required. In some embodiments, the liquid solution or suspension may be a consumable test sample (e.g., vegetable wash). In some embodiments, the liquid solution or suspension may be vegetable wash enriched with concentrated LB broth, Tryptic / Tryptone Soy broth, peptone water, or nutrient broth. In some embodiments, the liquid solution or suspension may be bacteria diluted in LB broth.

[0125] In some embodiments, lysis of the bacteria can occur before, during, or after the detection step. Experiments suggest that infected, unlysed cells may be detectable in some embodiments upon addition of luciferase substrate. Perhaps luciferase can leave the cells and / or luciferase substrate can enter the cells without complete cell lysis. Thus, for embodiments utilizing a spin filter system, lysis is required for detection if only the luciferase released into the lysate (and no luciferase in the intact bacteria) is analyzed in the luminometer. However, for embodiments utilizing a filter plate or 96-well plate with a solution or suspension sample, lysis is not essential for detection if the original plate filled with intact and lysed cells is assayed directly in the luminometer.

[0126] In some embodiments, the reaction between the indicator moiety (e.g., luciferase) and the substrate can continue for 30 minutes or longer, and detection at various time points can be desirable to optimize sensitivity. For example, in embodiments using a 96-well filter plate as the solid support and luciferase as the indicator, luminometer readings can be taken initially and at 10- or 15-minute intervals until the reaction is complete.

[0127] Surprisingly, the high concentrations of phage utilized to infect the test sample successfully achieved detection of very small numbers of target microorganisms in a very short time frame. Incubating the phage with the test sample, in some embodiments, only needs to be long enough for a single phage life cycle. In some embodiments, the concentration of bacteriophage for this incubation step is 7 x 10 6 , 8×10 6 , 9×10 6 , 1.0×10 7 , 1.1×10 7 , 1.2 × 107 , 1.3 × 10 7 , 1.4×10 7 , 1.5×10 7 , 1.6×10 7 , 1.7×10 7 , 1.8×10 7 , 1.9×10 7 , 2.0×10 7 , 3.0×10 7 , 4.0×10 7 , 5.0×10 7 , 6.0×10 7 , 7.0×10 7 , 8.0×10 7 , 9.0×10 7 , or 1.0×10 8 Higher than PFU / mL.

[0128] The success of phage at such high concentrations is surprising because many of the phage were previously associated with "non-infectious lysates," which killed target cells and thereby prevented the generation of useful signals from earlier phage assays. Purification of prepared phage stocks as described herein (e.g., clarification by cesium chloride isopycnic density gradient ultracentrifugation) is believed to help alleviate this problem because, in addition to removing any contaminating luciferase associated with the phage, this clarification can also remove ghost particles (particles that have lost their DNA). These ghost particles can lyse bacterial cells via "non-infectious lysates," prematurely killing the cells and thereby preventing the generation of indicator signals. Electron microscopy clearly shows that crude phage lysates (i.e., before cesium chloride clarification) can have more than 50% ghosts. These ghost particles may contribute to the early death of the microorganism through the action of many phage particles puncturing the cell membrane. Therefore, ghost particles may have contributed to previous problems where high PFU concentrations were reported to be harmful. Furthermore, the extremely clean phage preparation allows the assay to be performed without a washing step, which allows the assay to be performed without an initial enrichment step. Some embodiments include an initial enrichment step, which in some embodiments allows for a shorter enrichment incubation time.

[0129] Some embodiments of the test method may further include a confirmatory assay. A variety of assays are known in the art to confirm initial results, usually at a later time. For example, the sample may be cultured (e.g., CHROMAGAR®, DYNABEADS® assays described in the Examples), PCR may be used to confirm the presence of microbial DNA, or other confirmatory assays may be used to confirm the initial results.

[0130] In certain embodiments, the methods of the present invention may combine the use of a binding agent (e.g., an antibody) to purify and / or concentrate a microorganism of interest (e.g., Cronobacter spp.) from the sample in addition to detecting an infectious agent. For example, in certain embodiments, the present invention encompasses a method for detecting a microorganism of interest in a sample, comprising capturing the microorganism from the sample onto a support using a capture antibody specific for the microorganism of interest (e.g., Cronobacter spp.); incubating the sample with a recombinant bacteriophage that infects Cronobacter spp., wherein the recombinant bacteriophage contains an indicator gene inserted into the late gene region of the bacteriophage, such that expression of the indicator gene during bacteriophage replication after infection of a host bacterium results in a soluble indicator protein product; and detecting the indicator protein product, wherein positive detection of the indicator protein product indicates the presence of Cronobacter spp. in the sample.

[0131] For example, Figure 8 illustrates a hybrid immunophage (HIP) assay for detecting bacteria of interest using engineered bacteriophages according to one embodiment of the present invention. The samples are first applied to microtiter plate wells coated with bacteria-specific antibodies 802. The plate is then centrifuged to promote binding of the bacteria to the capture antibodies 804. After a time sufficient to allow complete bacterial capture, a solution containing bacteria-specific NANOLUC® phage is added to each sample 806. Incubation with the phage results in binding and attachment of single or multiple phages to the captured bacteria 808. Finally, the samples are incubated to promote phage replication and luciferase expression, which results in cell lysis and release of soluble luciferase 810.

[0132] In some embodiments, synthetic phage are engineered to optimize desired traits for use in pathogen detection assays. In some embodiments, bioinformatics and prior analysis of genetic modifications are used to optimize desired traits. For example, in some embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to specific species of bacteria. In other embodiments, genes encoding phage tail proteins can be optimized to recognize and bind to entire genuses of bacteria or specific groups of species within a genus. In this way, the phage can be optimized to detect broader or narrower groups of pathogens. In some embodiments, the synthetic phage can be engineered to improve expression of the reporter gene. Additionally and / or alternatively, in some cases, the synthetic phage can be engineered to increase the burst size of the phage to improve detection.

[0133] In some embodiments, the stability of the phage may be optimized to improve shelf life. For example, enzymatic solubility may be increased to increase subsequent phage stability. Additionally and / or alternatively, phage thermostability may be optimized. Thermostable phage better preserve functional activity during storage, thereby increasing shelf life. Thus, in some embodiments, the thermostability and / or pH tolerance may be optimized.

[0134] In some embodiments, the genetically modified phage or the synthetically derived phage comprises a detectable indicator. In some embodiments, the indicator is luciferase. In some embodiments, the phage genome comprises an indicator gene (e.g., a luciferase gene or another gene encoding a detectable indicator).

[0135] Systems and kits of the present invention In some embodiments, the present invention includes a system (e.g., an automated system or kit) comprising components for carrying out the methods disclosed herein. In some embodiments, a system or kit according to the present invention includes an indicator phage. The methods described herein may also utilize such an indicator phage system or kit. Some embodiments described herein are particularly suitable for automation or kits given the minimal amount of reagents and materials required to carry out the methods. In certain embodiments, each of the components of the kit may comprise a self-contained unit deliverable from a first site to a second site.

[0136] In some embodiments, the present invention includes a system or kit for rapid detection of a microorganism of interest in a sample. The system or kit may, in certain embodiments, include components for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent comprises an indicator moiety, and components for detecting the indicator moiety. In some embodiments of both the systems and kits of the present invention, the infectious agent is a recombinant bacteriophage that infects the bacterium of interest, and the recombinant bacteriophage comprises an indicator gene inserted into the bacteriophage's late gene region as the indicator moiety, such that expression of the indicator gene during bacteriophage replication after infection of the host bacterium results in a soluble indicator protein product. Some systems further include a component for capturing the microorganism of interest on a solid support.

[0137] In other embodiments, the present invention includes methods, systems, or kits for rapid detection of a microorganism of interest in a sample, comprising an infectious agent component specific for the microorganism of interest, wherein the infectious agent comprises an indicator moiety and a component for detecting the indicator moiety. In some embodiments, the bacteriophage is a T4-like, ViI, ViI-like, or Cronobacter spp.-specific bacteriophage. In one embodiment, the recombinant bacteriophage is derived from a Cronobacter spp.-specific bacteriophage. In certain embodiments, the recombinant bacteriophage is highly specific for a particular bacterium. For example, in certain embodiments, the recombinant bacteriophage is highly specific for Cronobacter spp. In embodiments, the recombinant bacteriophage can distinguish Cronobacter spp. in the presence of other types of bacteria. In certain embodiments, the system or kit detects as few as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 specific bacteria in the sample.

[0138] In certain embodiments, the system and / or kit may further include a component for washing the captured microbial sample. Additionally or alternatively, the system and / or kit may further include a component for determining the amount of the indicator moiety, where the amount of detected indicator moiety corresponds to the amount of microorganisms in the sample. For example, in certain embodiments, the system or kit may include a luminometer or other device for measuring luciferase enzyme activity.

[0139] In some systems and / or kits, the same components may be used for multiple steps. Thus, the steps are automated or controlled by a user via computer input and / or a liquid handling robot performs at least one step.

[0140] Thus, in certain embodiments, the present invention may include a system or kit for rapid detection of a microorganism of interest in a sample, the system or kit including: a component for incubating the sample with an infectious agent specific for the microorganism of interest, wherein the infectious agent comprises an indicator moiety; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microorganism sample to remove unbound infectious agent; and a component for detecting the indicator moiety. In some embodiments, the same component may be used for the capturing, incubating, and / or washing steps (e.g., a filter component). Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, wherein the amount of indicator moiety detected corresponds to the amount of the microorganism in the sample. Such a system may include various embodiments and subembodiments similar to those described above with respect to the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage. In a computerized system, the system may be fully automated, semi-automated, or directed by a user via a computer (or some combination of these).

[0141] In some embodiments, the system may include components for isolating the microorganism of interest from other components in the sample.

[0142] In one embodiment, the present invention encompasses a system or kit comprising components for detecting a microorganism of interest, the system comprising: a component for isolating at least one microorganism from other components in the sample; a component for infecting the at least one microorganism with multiple parent infectious agents; a component for lysing the at least one infected microorganism to release progeny infectious agents present in the microorganism; and a component for detecting the progeny infectious agents, or, with greater sensitivity, a component for detecting a soluble protein encoded and expressed by the infectious agent, wherein the infectious agent or a soluble protein product of the infectious agent indicates the presence of the microorganism in the sample. The infectious agent may comprise a Cronobacter-specific NANOLUC® bacteriophage carrying the NANOLUC® indicator gene.

[0143] The system or kit may include various components for detecting progeny infectious agents. For example, in one embodiment, the progeny infectious agent (e.g., bacteriophage) may include an indicator moiety. In one embodiment, the indicator moiety in the progeny infectious agent (e.g., bacteriophage) may be a detectable moiety (e.g., a soluble luciferase protein) expressed during replication.

[0144] In other embodiments, the present invention may include a kit for rapid detection of a microorganism of interest in a sample, the system including components for incubating the sample with an infectious agent specific for the microorganism of interest, where the infectious agent includes an indicator moiety; a component for capturing the microorganism from the sample onto a solid support; a component for washing the captured microorganism sample to remove unbound infectious agent; and a component for detecting the indicator moiety. In some embodiments, the same components may be used for the capturing, incubating, and / or washing steps. Some embodiments further include a component for determining the amount of the microorganism of interest in the sample, where the amount of indicator moiety detected corresponds to the amount of the microorganism in the sample. Such a kit may include various embodiments and subembodiments similar to those described above with respect to the method for rapid detection of a microorganism. In one embodiment, the microorganism is a bacterium and the infectious agent is a bacteriophage.

[0145] In some embodiments, kits may include components for isolating the microorganism of interest from other components in the sample.

[0146] These systems and kits of the present invention include various components. As used herein, the term "component" is broadly defined and includes any suitable device or collection of suitable devices for carrying out the described method. The components need not be integrally connected or mounted relative to one another in any particular way. The present invention encompasses any suitable arrangement of the components relative to one another. For example, the components need not reside in the same space. However, in some embodiments, the components are connected to one another in an integral unit. In some embodiments, the same component may perform multiple functions.

[0147] Computer System and Computer-Readable Medium The system may be embodied in the form of a computer system, as described in the present technology or any of its components. Representative examples of computer systems include general purpose computers, programmed microprocessors, microcontrollers, peripheral integrated circuit elements, and other devices or arrangements of devices capable of implementing the steps comprising the methods of the present technology.

[0148] The computer system may include a computer, an input device, a display unit, and / or the Internet. The computer may further include a microprocessor. The microprocessor may be connected to a communication bus. The computer may also include memory. The memory may include random access memory (RAM) and read-only memory (ROM). The computer system may further include a storage device. The storage device may be a hard disk drive or a removable storage device (e.g., a floppy disk drive, an optical disk drive, etc.). The storage device may also be other similar means for loading computer programs or other instructions into the computer system. The computer system may also include a communication unit. The communication unit allows the computer to connect to other databases and the Internet through an I / O interface. The communication unit allows data to be transferred to and received from other databases. The communication unit may include a modem, an Ethernet card, or any similar device that allows the computer system to connect to databases and networks (e.g., LANs, MANs, WANs, and the Internet). The computer system may therefore facilitate input from a user through input devices accessible to the system via an I / O interface.

[0149] A computing device typically includes an operating system that provides executable program instructions for the general management and operation of the computing device, and typically includes a computer-readable storage medium (e.g., a hard disk, random access memory, read-only memory, etc.) that stores instructions that, when executed by a server's processor, cause the computing device to perform its intended functions. Suitable implementations of such operating systems and the general functionality of such computing devices are known or commercially available and are readily implemented by those skilled in the art, especially in light of the disclosure herein.

[0150] The computer system executes a set of instructions stored in one or more memory devices to process input data. The memory devices may also hold data or other information as described. The memory devices may be in the form of information sources or physical memory devices present in a processing machine.

[0151] The environment may include various data stores and other memory and storage media, as discussed above. These may reside in a variety of locations (e.g., in storage media local to (and / or residing in) one or more of the computers, or remote from any or all of the computers across a network). In a particular group of embodiments, information may reside on a storage area network ("SAN"), familiar to those skilled in the art. Similarly, any files necessary to perform the functions ascribed to the computers, servers, or other network devices may be stored locally and / or remotely, as appropriate. Where the system includes computing devices, each such device may include hardware elements that may be electrically coupled via a bus, including, for example, at least one central processing unit (CPU), at least one input device (e.g., a mouse, keyboard, controller, touchscreen, or keypad), and at least one output device (e.g., a display device, printer, or speaker). Such a system may also include one or more storage devices (e.g., disk drives, optical storage devices, and solid-state storage devices such as random access memory ("RAM") or read-only memory ("ROM"), as well as removable media devices, memory cards, flash cards, etc.

[0152] Such devices may also include a computer-readable storage medium reader, a communication device (e.g., a modem, a network card (wireless or wired), an infrared communication device, etc.), and a working memory as described above. The computer-readable storage medium reader may be connected to or configured to accept computer-readable storage media representing remote, local, fixed, and / or removable storage devices, as well as storage media for temporarily and / or more permanently containing, storing, transmitting, and retrieving computer-readable information. The systems and various devices also typically include many software applications, modules, services, or other elements located within at least one working memory device, including an operating system and application programs (e.g., client applications or web browsers). It should be recognized that alternative embodiments may have many variations from those described above. For example, customized hardware may also be used, and / or particular elements may be implemented in hardware, software (including portable software (e.g., applets)), or both. Additionally, connections to other computing devices (e.g., network input / output devices) may be used.

[0153] Non-transitory storage media and computer-readable media for containing code or portions of code may include any suitable media known or used in the art, including storage media and communication media (e.g., volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage and / or transmission of information such as computer-readable instructions, data structures, program modules, or other data), including RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital versatile disk (DVD) or other optical storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage device, or any other medium that can be used to store the desired information and that can be accessed by the system device. Based on the above disclosure and the teachings provided herein, one skilled in the art will recognize other ways and / or methods to implement the various embodiments.

[0154] Computer-readable media may include, but are not limited to, electronic, optical, magnetic, or other storage devices capable of providing computer-readable instructions to a processor. Other examples include, but are not limited to, floppy disks, CD-ROMs, DVDs, magnetic disks, memory chips, ROM, RAM, SRAM, DRAM, content addressable memory ("CAM"), DDR, flash memory (e.g., NAND flash or NOR flash), ASICs, configured processors, optical storage media, magnetic tape or other magnetic storage media, or any other medium from which a computer processor can read instructions. In one embodiment, the computing device may include a single type of computer-readable medium (e.g., random access memory (RAM)). In other embodiments, the computing device may include two or more types of computer-readable media (e.g., random access memory (RAM), disk drives, and cache). The computing device may be in communication with one or more external computer-readable media (e.g., an external hard disk drive, or an external DVD or Blu-ray drive).

[0155] As discussed above, the embodiments include a processor configured to execute computer-executable program instructions and / or access information stored in memory. The instructions may include processor-specific instructions generated by a compiler and / or interpreter from code written in any suitable computer programming language, including, for example, C, C++, C#, Visual Basic, Java, Python, Perl, JavaScript, and ActionScript (Adobe Systems, Mountain View, Calif.). In one embodiment, the computing device includes a single processor. In other embodiments, the device includes two or more processors. Such processors may include microprocessors, digital signal processors (DSPs), application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), and state machines. Such processors may include programmable electronic devices (e.g., PLCs), programmable interrupt controllers (PICs), programmable logic devices (PLDs), programmable read-only memories (PROMs), electronically programmable read-only memories (EPROMs or EEPROMs), and the like. or other similar devices.

[0156] The computing device includes a network interface. In some embodiments, the network interface is configured to communicate over a wired or wireless communication link. For example, the network interface may enable communication over a network via Ethernet, IEEE 802.11 (Wi-Fi), 802.16 (Wi-Max), Bluetooth, infrared, etc. As another example, the network interface may enable communication over a network (e.g., CDMA, GSM, UMTS, or other cellular communication network). In some embodiments, the network interface may enable communication over a network with another device, e.g., a Universal Serial Bus (USB), 1394 It may allow point-to-point connections via FireWire, serial or parallel connections, or similar interfaces. Some embodiments of suitable computing devices may include two or more network interfaces for communication over one or more networks. In some embodiments, the computing device may include a data store in addition to or instead of a network interface.

[0157] Some embodiments of a suitable computing device may include or be in communication with a number of external or internal devices (e.g., a mouse, a CD-ROM, a DVD, a keyboard, a display, an audio speaker, one or more microphones, or any other input or output device). For example, the computing device may be in communication with a variety of user interface devices and displays. The displays may use any suitable technology, including, but not limited to, LCD, LED, CRT, etc.

[0158] The set of instructions for execution by the computer system may include various commands that instruct a processing machine to perform particular tasks (e.g., steps constituting the methods of the present technology). The set of instructions may be in the form of a software program. Moreover, the software may be in the form of a collection of separate programs, a program module with a larger program, or a portion of a program module, as in the present technology. The software may also include modular programming in the form of object-oriented programming. The processing of input data by the processing machine may be in response to user commands, results of previous processing, or a request made by another processing machine.

[0159] While the present invention has been disclosed with reference to certain embodiments, many modifications, changes, and variations to the described embodiments are possible without departing from the scope and spirit of the invention, as defined in the appended claims. Accordingly, the present invention is not intended to be limited to the above-described embodiments, but has full scope defined by the language of the following claims and equivalents thereof. [Example]

[0160] The results presented in the Examples below demonstrate detection of low numbers of cells, even single bacteria, with reduced time to results.

[0161] Example 1. Generation and isolation of indicator phages from Cronobacter-specific bacteriophages Indicator phage Cronobacter-specific Saka2.NANOLUC, Saka4.NANOLUC, and Saka10.NANOLUC bacteriophages were generated through homologous recombination using procedures as previously described. Cronobacter phages Saka2, Saka4, and Saka10 were isolated from sewage samples.

[0162] The genome sequences of these phages were obtained through whole-genome sequencing using the Illumina MiSeq system with de novo sequence assembly. Based on previously known and annotated genomes of related phages, the late gene regions and major capsid protein genes were located on the new phage genomes. Plasmids were designed and synthesized to insert NANOLUC® with appropriate late gene promoters and ribosome binding sites flanked by approximately 500 bp of matching phage sequence to facilitate homologous recombination.

[0163] Target bacteria were transformed with the homologous recombination plasmids under appropriate antibiotic selection and infected with their respective wild-type phages to allow homologous recombination with the plasmids. After homologous recombination to generate the recombinant bacteriophage genomes, a series of titration and enrichment steps were used to isolate specific recombinant bacteriophages expressing NANOLUC® as previously described.

[0164] Finally, large-scale production was performed to obtain high-titer stocks suitable for use in Cronobacter spp. detection assays. Cesium chloride isopycnic density gradient centrifugation was used to separate phage particles from contaminating luciferase protein and reduce background.

[0165] Example 2. Infant Formula Protocol - Cronobacter A Cronobacter bacterial suspension was grown overnight in medium. After overnight incubation, the culture was diluted to the desired concentration in sterile water. The diluted cells were plated on non-selective plates to quantitate CFU. Powdered infant formula (PIF) was spiked with Cronobacter dilutions. The inoculated PIF was dried at a high heat setting using a vacuum pump. The dried and spiked PIF samples were then used to inoculate 10g, 100g, or 300g samples of PIF at various CFU levels. The inoculated PIF samples were stored at room temperature for 2-4 weeks before evaluation.

[0166] 10 g, 100 g, and 300 g samples of PIF were prepared. A 10 g PIF sample was diluted 1:9 with 90 mL of pre-warmed (37°C) buffered peptone water (BPW) medium. The samples were mixed at a sample:volume ratio; 100 g of PIF was mixed with 300 mL of pre-warmed BPW medium at a 1:3 sample:volume ratio; and 300 g of PIF was mixed with 900 mL of pre-warmed BPW medium at a 1:3 sample:volume ratio. Using a STOMACHER® or equivalent device (peristaltic blender), the samples were homogenized at the highest setting for 120 seconds. The homogenized samples were incubated at 37°C without shaking for 16-18 hours. After incubation, the samples were mixed thoroughly, and 1 mL of sample was removed and transferred to a centrifuge tube. The samples were diluted 1:10 in BPW (100 μl sample: 900 μl BPW). 150 μl of the diluted samples were transferred to a 96-well plate. Ten microliters of a bacteriophage cocktail of Cronobacter-specific bacteriophage solution was added to each well and incubated for 2 hours at 37°C. Ten microliters of lysis buffer was added to each well, followed by 50 μl of prepared luciferase substrate and read on a luminometer. Samples positive for Cronobacter had a reading of 500 or greater relative light units (RLU), and negative samples had a reading of less than 500 RLU.

[0167] Positive detection of Cronobacter was confirmed. Samples were enriched for 24 hours at 37°C. 50 μl of each enriched sample was plated onto a Brilliance plate (Oxoid TM Brilliance TM Cronobacter sakazakii (Agar cat# OXCM0129R) was streaked onto the culture and incubated at 37°C for 24 hours. Positive detection was confirmed by blue-green colony growth. [Table 1]

[0168] 100g PIF samples inoculated at different levels and with different Cronobacter strains (Table 1) were tested for Cronobacter using the infant formula assay. For the 100g samples, a 1:3 dilution of PIF to BPW (buffered peptone water) was used. Samples with a reading greater than 500 RLU / s or 5:1 signal / background were considered positive. 24-hour enrichment samples were plated on Brilliance plates for confirmation.

[0169] The matrices tested included: infant formula (milk-based), infant formula (soy-based), infant formula supplemented with prebiotics and probiotics, infant formula with added rice starch, or skim milk powder.

[0170] Example 3. Inclusion and Exclusion Study Inclusive strains (Cronobacter) were obtained from academic, government, and commercially available sources (Table 2). Each strain was grown overnight in TSB medium at 37±1°C to stationary phase. Cells were diluted to 100 CFU in 0.1 mL of TSB and mixed with recombinant phage for 2 hours at 37±1°C. After infection, samples were mixed with lysis buffer and luciferase substrate and then read in a luminometer. Samples with RLU values ​​greater than 150 were considered positive. Excluding strains were also obtained from commercially available sources and grown overnight to stationary phase. Assays with excluding strains were performed as with the inclusive strains, except that the overnight cultures were assayed directly (Table 3).

[0171] The Cronobacter assay shows 100% inclusion of 75 Cronobacter strains tested (Table 2). The Cronobacter assay also shows exclusion of 38 of 41 non-Cronobacter strains tested (Table 3). The three non-Cronobacter strains detected by the Cronobacter assay were from the closely related Enterobacter genus. Any putative positives from the phage assay can be enriched for a total of 24±2 hours at 37±1°C and then treated with OXOID. TM BRILLIANCE TM Cronobacter Sakazakii may be streaked onto Agar and further incubated for 24±2 hours at 37±1° C. for confirmation. The presence of colonies that are well grown (1 mm to 3 mm) and appear blue-green indicates a positive sample. [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2]

[0172] Example 4. Strength studies Three parameters were varied to demonstrate assay strength: enrichment time (14 and 24 hours), recombinant phage concentration (±20%), and luciferase substrate amount (±10%). Briefly, 10 g milk-based infant formula samples were either left unspiked or spiked with 0.2-2 CFU / 10 g C. muytjensii FSL-F6-031 dried in infant formula and stored at room temperature (20-25°C) for 2-4 weeks. The Cronobacter assay protocol described above was followed, with variations in enrichment time, recombinant phage concentration, and substrate amount as shown in Table 4. Samples with RLU values ​​greater than 500 were considered positive. Samples were enriched for a total of 24±2 hours, then spiked with OXOID. TM BRILLIANCE TM Cronobacter Samples were confirmed by plating on Sakazakii Agar. Plates were incubated for an additional 24±2 hours at 37±1°C. The presence of well-grown blue-green colonies indicated a positive sample. A summary of the above tests is shown in Table 4.

[0173] Robustness testing of the Cronobacter assay showed that variations in enrichment time, recombinant phage concentration, and luciferase substrate amount did not alter the results compared to the standard protocol. Enrichment times of 14 and 24 hours, recombinant phage volumes of 8 and 12 μL, and luciferase substrate volumes of 45 and 55 μL produced identical results to the standard protocol of 16 hours of enrichment, 10 μL of recombinant phage, and 50 μL of luciferase substrate in both the uninoculated and low-inoculum test samples (Table 4). These results indicate that these deviations from the Cronobacter assay protocol did not alter the final results. [Table 4]

[0174] Example 5. Matrix study A matrix study compared Cronobacter (10 g test portion) with ISO 22964:2006 (10 g test portion) and Cronobacter (100 g and 300 g test portions) with FDA BAM Ch. 29 Cronobacter:2012 (100 g test portion). The Cronobacter 10 g portion was compared with ISO 22964:2006 using a paired study design. The Cronobacter 100 g and 300 g portions were compared with the FDA BAM Ch. 29 100 g portion using an unpaired study design. For each matrix and each comparison, the study included five replicate test portions of uninoculated matrix (0 CFU / test portion), 20 replicate test portions at low levels to obtain fractionally positive results (0.2-2 CFU / test portion), and five replicate test portions at high levels to obtain consistently positive results (2-10 CFU / test portion).

[0175] Both milk- and soy-based PIF were purchased from local retailers and prescreened for common contamination using the ISO 22964:2006 method. To prepare the inoculum, Cronobacter was grown in tryptic soy broth for 18-24 hours at 37 ± 1°C. The culture was diluted in BPW, reconstituted in PIF, and placed in a speed vacuum for 4-8 hours until the sample was completely dry. After drying, the dried inoculum was diluted into the PIF matrix used in each study to obtain low levels expected to yield graded positive results and high levels expected to yield total positive results, and allowed to stand at room temperature (20-25°C) for 2-4 weeks to allow equilibration in the matrix. Bulk lots of the matrix were inoculated with the diluted inoculum prior to testing.

[0176] On the day of analysis, total aerobic counts were determined according to FDA BAM Ch. 3, and Cronobacter levels in the low- and high-level inocula were determined by most-probable-number (MPN) analysis. For paired samples, MPN analysis was determined using the ISO 22694:2006 method. For the low-level inocula, five test portions of 25 g, five test portions of 4 g, and 20 test portions of 10 g from the matrix study were analyzed. For the high-level inocula, five test portions of 10 g, five test portions of 4 g, and five test portions of 1.5 g from the matrix study were analyzed.

[0177] For unpaired samples, MPN analysis was determined using the FDA BAM Ch. 29 method. For the low-level inoculum, five test portions of 200 g, five test portions of 50 g, and twenty test portions of 100 g from the matrix study were analyzed. For the high-level inoculum, five test portions of 100 g, five test portions of 50 g, and five test portions of 25 g from the matrix study were analyzed. The number of positives was used to calculate the MPN using the LCF MPN calculator provided by the AOAC RI.

[0178] The Cronobacter test portions were processed according to the manufacturer's instructions. Briefly, 90 mL (10 g test portion), 300 mL (100 g test portion) of pre-warmed BPW (37 ± 1°C) were added. 900 mL (300 g test portion) or 900 mL (300 g test portion) of PIF was added to the PIF test portion. The samples were homogenized and enriched at 37 ± 1°C for 16–18 hours. The enriched samples were mixed thoroughly, and then aliquots were taken for analysis. The samples were diluted 1:10 (100 μl sample: 900 μl BPW) in pre-warmed BPW (37 ± 1°C), and 150 μl of the diluted sample was transferred to a 96-well plate. The samples were then infected with recombinant phage for 2 hours at 37 ± 1°C. Lysis buffer and luciferase substrate were added to the samples. The samples were then read in a luminometer. A reading of ≥ 500 RLU was considered positive. To confirm the Cronobacter assay, samples were enriched for a total of 24 ± 2 hours at 37 ± 1°C. The enriched sample was mixed thoroughly, after which an aliquot was taken for analysis. 50 μL was added to the OXOID TM BRILLIANCE TM Cronobacter Sakazakii was infiltrated onto agar and incubated for 24±2 hours at 37±1° C. The presence of colonies that grew well (1 mm to 3 mm) and appeared blue-green indicated a positive sample.

[0179] For confirmation per FDA BAM Ch. 29, sections E and F were performed. Briefly, two 40 mL aliquots from the 24-hour enrichment were centrifuged at 3,000 × g for 10 minutes. The supernatant was discarded, and the resulting pellet was resuspended in 200 μl of sterile phosphate-buffered saline. 100 μl aliquots of the resuspended pellet were plated onto two DFI chromogenic agar plates and two R&F Cronobacter chromogenic agar plates. Additionally, one loopful of each enrichment was infiltrated onto two DFI chromogenic agar plates and two R&F Cronobacter chromogenic agar plates. All plates were incubated at 36 ± 1°C for 18–24 hours. Putative positive colonies were confirmed by PCR as outlined in section F of BAM Ch. 29.

[0180] ISO 22964:2006 (current version at the time of testing) was used in the method developer's laboratory for matrix evaluation. Briefly, 90 mL of BPW was added to 10 g of PIF. The sample was incubated at 37 ± 1°C for 18 ± 2 hours. 0.1 mL was then transferred from the BPW culture to 10 mL of mLST / vancomycin medium and incubated at 44 ± 1°C for 24 ± 2 hours. One loopful of mLST / vancomycin culture was inoculated onto Enterobacter sakazakii Isolation Agar and incubated at 44 ± 1°C for 24 ± 2 hours. One to five presumptive positive colonies were then inoculated onto tryptic soy agar (TSA) plates and incubated at 25°C for 48 ± 4 hours. Yellow-stained colonies were selected for further biochemical confirmation testing.

[0181] For the FDA BAM Ch. 29 Cronobacter method, 900 mL of sterile BPW was added to 100 g PIF in a sterile 2 L Erlenmeyer flask and gently stirred by hand until the PIF was uniformly suspended. Test samples were incubated at 36 ± 1°C for 24 ± 2 hours. After enrichment, the samples were thoroughly mixed, and 4 × 40 mL aliquots from each sample were transferred to 50 mL centrifuge tubes. The aliquots were centrifuged at 3,000 × g for 10 minutes, and the supernatant was discarded. The resulting pellet was resuspended in 200 μl of phosphate-buffered saline. Two aliquots were used for PCR to determine presumptive positives, and two aliquots were used for culture confirmation, if necessary. For the PCR screen, two aliquots were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 3,000 × g for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 400 μL of PREPMAN ULTRA® Sample Preparation Reagent and vortexed at maximum speed until the pellet was completely resuspended. The samples were heated to 100°C in a dry bath incubator for 10 minutes and then cooled to room temperature. Once the samples reached room temperature, they were centrifuged at 15,000 × g for 2 minutes, and a 50 μL aliquot of the supernatant was transferred to a new microcentrifuge tube for PCR analysis. For each sample, PCR analysis was performed with or without an internal control (InC). The PCR reaction components and the PCR protocol were followed as outlined in the FDA BAM Chapter 29 reference method. Presumptive positives were confirmed using FDA BAM Ch. 29, Sections E and F. Briefly, 100 μL aliquots of the resuspended pellet were plated onto two DFI chromogenic agar plates and two R&F Cronobacter chromogenic agar plates. Additionally, one loopful of each enrichment was streaked onto two DFI chromogenic agar plates and two R&F Cronobacter chromogenic agar plates. All plates were incubated at 36 ± 1°C for 18–24 hours. Colonies were confirmed by PCR as outlined in Section F of BAM Ch. 29.

[0182] All study results were analyzed using POD statistical analysis with 95% confidence intervals (CI). POD analysis is described in the AOAC INTERNATIONAL guidelines in Appendix J. Data from the above analyses are presented in Tables 5-8.

[0183] The method developer study demonstrated no differences between the assay and the ISO 22964:2006 and FDA BAM Ch. 29 Cronobacter reference methods for all matrices tested (Tables 5–8). All test fractions that were presumptively positive by the Cronobacter assay were confirmed to contain Cronobacter by their respective reference methods. There were no false-negative results. The POD analysis demonstrated no significant differences between the Cronobacter assay and the ISO 22964:2006 reference method in paired studies (Table 5). There was no statistical difference between the number of Cronobacter assay presumptive and BAM Ch. 29 confirmed results (Table 6). Comparisons of Cronobacter assay results and BAM Ch. 29 confirmatory, as well as presumptive and confirmed results, were also not statistically significant (Tables 6 and 7). Comparisons of the Cronobacter assay and FDA BAM Ch. 29 unpaired studies demonstrated no statistical differences in the performance of the two methods (Table 8). The one exception was the 100g milk-based assay vs. BAM Ch. 29 method comparison (Table 8). The difference between the graded positives was statistically significant, with a dPOD of 0.35 and a CI of (0.04, 0.58). The aerobic bacterial plate count of the PIF used in the study was 0 CFU / g, indicating that the PIF had no or very low levels of background flora present at the start of the enrichment process.

[0184] The independent experimental evaluation included a matrix study comparing the Cronobacter assay to ISO 22964:2017 and FDA BAM Chapter 29 reference methods for milk-based PIF. For the method comparison to ISO 22964:2017, 30 paired 10g test portions were evaluated. For the method comparison to FDA BAM Chapter 29, 100g and 300g test portions of the Cronobacter assay were compared to 100g test portions of the reference method. Within each sample set, there were five uninoculated samples (0 CFU / test portion), 20 low-inoculated samples (0.2-2 CFU / test portion), and five high-inoculated samples (2-10 CFU / test portion). The low inoculation levels were designed to generate graded positive results (the candidate or reference method would produce 5-15 positive results (25-75%)).

[0185] The PIF was purchased from a local distributor, prescreened for routine analyte contamination according to ISO 22964:2017, and analyzed for total aerobic bacterial counts according to FDA BAM Chapter 3. After screening, the matrix was inoculated with a strain of Cronobacter species. For validation, a lyophilized culture was used to inoculate the PIF. The lyophilized culture was prepared by transferring a single Cronobacter sakazakii colony from tryptic soy agar containing 5% sheep blood into brain heart infusion (BHI) broth and incubating the culture at 35 ± 2°C for 18–24 hours. After incubation, the culture was diluted in sterile cryoprotectant (reconstituted nonfat dry milk) (NFDM) and placed on a lyophilization system for 48–72 hours. After removing the culture from the lyophilization system, the lyophilized culture was diluted in NFDM to a low level expected to produce graded positive results and a high level expected to produce a total positive result. A bulk lot of the matrix was inoculated. After inoculation, the matrix was kept at room temperature (24±2° C.) for two weeks to allow equilibration of the organisms in the matrix.

[0186] Total aerobic bacterial counts were determined according to FDA BAM Ch. 3. Cronobacter levels in the low-level and high-level inocula were determined by MPN on the day of analysis. For paired sample analysis, the low-level MPN was determined for 5 x 25 g samples. The levels of Cronobacter in the high level inoculum were determined by evaluating 5 x 10 g test portions, 5 x 4 g test portions, and 5 x 1.5 g test portions from the above study.

[0187] For unpaired analyses, the low-level MPN was determined by evaluating 5 x 200 g test portions, 20 x 100 g reference method test portions, and 5 x 50 g test portions from the study. Cronobacter levels in the high-level inoculum were determined by evaluating 5 x 100 g reference method test portions, 5 x 50 g test portions, and 5 x 25 g test portions from the study. Each test portion was enriched with BPW and analyzed by the reference method procedure. The number of positives from the three test levels was used to calculate the MPN using the LCF MPN calculator (version 1.6).

[0188] For ISO 22964:2017, a 10 g PIF test portion was enriched with 90 mL of BPW (ISO formulation) and incubated at 37 ± 1°C for 18 ± 2 hours. After incubation, 0.1 mL of the primary enrichment was transferred to 10 mL of Cronobacter selective broth (CSB) and incubated at 41.5 ± 1°C for 24 ± 2 hours. After incubation, one loopful of the CSB was infiltrated onto Chromogenic Cronobacter Isolation (CCI) agar and incubated at 41.5 ± 1°C for 24 ± 2 hours. After incubation of the CCI plate, one to five colonies of representative Cronobacter species (medium-sized, 1 mm to 3 mm, blue-green to blue colonies) were transferred to tryptone soy agar (TSA) and incubated at 35 ± 1°C for 18 to 24 hours. After incubation, representative colonies (1-3 mm yellow-stained) were subjected to an oxidase test, and final biochemical confirmation was performed using VITEK® 2 GN Biochemical Identification cards according to AOAC Official Method 2011.17.

[0189] For BAM Ch. 29, a 100 g PIF test portion was added to a 2 L Erlenmeyer flask, enriched with 900 mL of prewarmed (37°C) BPW, and incubated at 37°C ± 1°C for 24°C ± 2 hours. After incubation, four 40 mL aliquots were transferred to four 50 mL conical vials. The aliquots were centrifuged at 3,000 x g for 10 minutes. For each conical tube, the supernatant was aspirated, and the lipid precipitate was removed using a sterile cotton swab. The remaining pellet was resuspended by adding 200 μL of phosphate-buffered saline and vortexing the suspension at maximum speed for 20 seconds. For each sample, two of the aliquots were used for PCR screening of Cronobacter, and two of the aliquots were used for culture confirmation.

[0190] For PCR screening, two aliquots were transferred to separate 1.5 mL microcentrifuge tubes and centrifuged at 3,000 × g for 5 minutes. The supernatant and lipid layer were removed, and the pellet was resuspended by adding 400 μL of PREPMAN ULTRA® Sample Preparation Reagent and vortexing at maximum speed until suspension was achieved. The samples were heat-treated at 100°C for 10 minutes in a dry bath incubator and then cooled to room temperature. Once the samples reached room temperature, they were centrifuged at 15,000 × g for 2 minutes, and a 50 μL aliquot of the supernatant was transferred to a new microcentrifuge tube for PCR analysis. For each sample, PCR analysis was performed with and without an internal control (InC). The PCR reaction components and PCR protocol were followed as outlined in the FDA BAM Ch. 29 reference method.

[0191] Regardless of the presumptive PCR results, 100 μL of suspended cells from each sample was infiltrated onto two DFI chromogenic agar plates and two R&F agar plates. The DFI chromogenic agar plates and R&F agar plates were incubated at 36 ± 1°C for 18–24 hours. After incubation, representative Cronobacter colonies from the DFI chromogenic agar plates (light to dark green, brownish, or green centers with white to yellow edges) and from the R&F agar plates (red, blue to black, or blue to gray backgrounds) were biochemically confirmed using VITEK® 2 GN Biochemical Identification Cards (AOAC Official Method 2011.17) and PCR analysis.

[0192] For all three species levels, POD analysis between the Cronobacter assay and the reference method showed that there was no statistically significant difference at the 5% level between the number of positive results obtained by the methods (Tables 5-8). For all three species levels, POD analysis between the estimated and confirmed results of the Cronobacter assay was at the 5% level for all test portions analyzed. The aerobic bacterial plate count of the PIF used in the study was 40 CFU / g, which indicates that the PIF could produce approximately 400 CFU (10 g), 4,000 CFU (100 g), or 12,000 CFU (100 g). CFU (300 g) of background bacterial flora present at the start of the entire process. [Table 5] [Table 6] [Table 7] [Table 8] The present invention provides, for example, the following items. (Item 1) A recombinant bacteriophage comprising an indicator gene inserted into the late gene region of the bacteriophage genome, wherein said recombinant bacteriophage specifically infects Cronobacter spp. (Item 2) 2. The recombinant bacteriophage according to item 1, wherein the recombinant bacteriophage is derived from a wild-type Saka2, Saka4, or Saka10 bacteriophage. (Item 3) 2. The recombinant bacteriophage of item 1, wherein the indicator gene is codon-optimized and encodes a soluble protein product that generates an endogenous signal or a soluble enzyme that generates a signal upon reaction with a substrate. (Item 4) 2. The recombinant bacteriophage according to item 1, further comprising an untranslated region upstream of the codon-optimized indicator gene, the untranslated region comprising a bacteriophage late gene promoter and a ribosome entry site. (Item 5) A cocktail composition comprising at least two different types of recombinant bacteriophages, wherein at least one of the recombinant bacteriophages comprises the indicator gene described in item 1. (Item 6) 1. A method for preparing a recombinant indicator bacteriophage, comprising: selecting a wild-type bacteriophage that specifically infects a target pathogenic bacterium; preparing a homologous recombination plasmid / vector containing an indicator gene; transforming the homologous recombination plasmid / vector into the target pathogenic bacterium; infecting the transformed target pathogenic bacterium with the selected wild-type bacteriophage, thereby allowing homologous recombination to occur between the plasmid / vector and the genome of the bacteriophage; and Isolating specific clones of recombinant bacteriophage A method comprising: (Item 7) The step of preparing a homologous recombination plasmid / vector includes: determining the native nucleotide sequence in the late region of the genome of said selected bacteriophage; annotating the genome and identifying the major capsid protein gene of the selected bacteriophage; designing a sequence for homologous recombination downstream of the major capsid protein gene, the sequence comprising a codon-optimized indicator gene; and Incorporating said sequences into a plasmid / vector designed for homologous recombination Item 7. The method according to item 6, comprising: (Item 8) 8. The method of claim 7, wherein designing the sequence further comprises inserting an untranslated region comprising a phage late gene promoter and a ribosome entry site upstream of the codon-optimized indicator gene. (Item 9) 9. The method of claim 8, wherein the homologous recombination plasmid comprises a bacteriophage late gene promoter and an untranslated region comprising a ribosome entry site upstream of the codon-optimized indicator gene. (Item 10) 9. The method according to item 8, wherein the wild-type bacteriophage is a Cronobacter-specific bacteriophage and the target pathogenic bacterium is a Cronobacter spp. (Item 11) 9. The method according to item 8, wherein the step of isolating specific clones of recombinant bacteriophage comprises a limiting dilution assay to isolate clones that exhibit expression of the indicator gene. (Item 12) 1. A method for detecting Cronobacter spp. in a sample, the method comprising: incubating the sample with a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage containing an indicator gene inserted into the late gene region of the bacteriophage genome; and detecting an indicator protein product produced by the recombinant bacteriophage, wherein positive detection of the indicator protein product indicates that Cronobacter spp. is present in the sample; A method comprising: (Item 13) 13. The method of claim 12, wherein the sample is a food sample, an environmental sample, a water sample or a commercial sample. (Item 14) 13. The method of claim 12, which detects as few as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bacteria in a sample of standard size for the food safety industry. (Item 15) 15. The method of claim 14, wherein the food sample comprises meat, fish, vegetables, eggs, dairy products, dry food products, or infant formula. (Item 16) Item 17. The method of Item 12, wherein the sample is incubated with a cocktail composition containing at least two different types of recombinant bacteriophage, at least one of the recombinant bacteriophage containing the indicator gene of Item 12. 17. The method of claim 16, wherein the sample is initially incubated in conditions that favor growth for an enrichment period of less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. (Item 18) 17. The method of claim 16, wherein the total time to results is less than 26 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than 21 hours, less than 20 hours, less than 19 hours, less than 18 hours, less than 17 hours, less than 16 hours, less than 15 hours, less than 14 hours, less than 13 hours, less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, or less than 2 hours. (Item 19) 13. The method of claim 12, wherein the signal-to-background ratio produced by the step of detecting the indicator is at least 2.0 or at least 2.5. (Item 20) A kit for detecting Cronobacter spp., comprising a recombinant bacteriophage derived from a Cronobacter-specific bacteriophage. (Item 21) 21. The kit of claim 20, further comprising a substrate for reacting with an indicator to detect a soluble protein product expressed by the recombinant bacteriophage. (Item 22) A system for detecting Cronobacter spp. comprising a recombinant bacteriophage derived from a Cronobacter -specific bacteriophage.

Claims

[Claim 1] The invention as described in the drawings.

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